Compositions and methods for targeting stromal cells for the treatment of cancer

ABSTRACT

The present invention provides compositions and methods for treating cancer in a human. The invention relates to targeting the stromal cell population in a tumor microenvironment. For example, in one embodiment, the invention provides a composition that is targeted to fibroblast activation protein (FAP). The invention includes a chimeric antigen receptor (CAR) which comprises an anti-FAP domain, a transmembrane domain, and a CD3zeta signaling domain.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 14/042,306, filed Sep. 30, 2013, allowed, which is entitled topriority under 35 U.S.C. § 119(e) to U.S. Provisional Patent ApplicationNo. 61/708,336, filed Oct. 1, 2012, which is hereby incorporated byreference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under CA141144, RO1 CA172921, and P01 CA66726 awarded by the National Institutes of Health(NIH). The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Adoptive transfer of T cells directed against antigens expressed byneoplastic cells is an immunotherapeutic approach that has proveneffective in some patients. A recent advance in adoptive T cell therapy(ATCT) involves the use of T cells transduced with chimeric antigenreceptors (CARs) directed against tumor cell associated antigens.

CARs are typically engineered to contain three regions. The N-terminalextracellular region dictates the antigen specificity of CARs and isencoded by a single chain fragment variable region (scFv) derived fromthe linked V_(H) and V_(L) domains of the antigen binding region of amonoclonal antibody (mAb) specific for the intended targeted antigen.This ligand binding component is followed by a flexible hinge sequence,derived from a CD8α or immunoglobulin sequence and one or moreintracellular signaling domains, which may be derived from thecytoplasmic domains of TCR CD3-ε, CD3-γ, or CD3-ζ chains orhigh-affinity receptor for IgE (FcεRI). The main advantage of CARtechnology is that it combines the effector functions of T lymph ocyteswith the ability of antibodies to specifically bind antigens with highaffinity in a non-MHC restricted fashion. Furthermore, patient-derivedblood lymphocytes can be readily expanded and transduced with thedesired CAR.

A key need for ATCT is target molecules that will be specific to tumorsand effectively reduce tumor size when targeted. While most efforts havefocused on targeting tumor antigens, it is evident that other componentsof the tumor microenvironment, including stromal cells, infiltratingimmune cells, vasculature, and extracellular matrix, promote tumorgrowth and metastasis. These components therefore may representadditional therapeutic targets in order to minimize or eliminatecancerous tumors.

Thus, there is an urgent need in the art for compositions and methods totarget stromal cells for the treatment of cancer. The present inventionaddresses this need.

BRIEF SUMMARY OF THE INVENTION

The invention includes an isolated nucleic acid sequence encoding achimeric antigen receptor (CAR), wherein the CAR comprises an antigenbinding domain, a transmembrane domain, a costimulatory signalingregion, and a CD3 zeta signaling domain, wherein the antigen bindingdomain binds to a stromal cell antigen.

The invention further includes an isolated chimeric antigen receptor(CAR) comprising an antigen binding domain, a transmembrane domain, acostimulatory signaling region, and a CD3 zeta signaling domain, whereinthe antigen binding domain binds to a stromal cell antigen.

Also included in the invention is a cell comprising a nucleic acidsequence encoding a chimeric antigen receptor (CAR), the CAR comprisingan antigen binding domain, a transmembrane domain, a costimulatorysignaling region, and a CD3 zeta signaling domain, wherein the antigenbinding domain binds to a stromal cell antigen.

Further included is a vector comprising a nucleic acid sequence encodinga chimeric antigen receptor (CAR), wherein the CAR comprises an antigenbinding domain, a transmembrane domain, a costimulatory signalingregion, and a CD3 zeta signaling domain, wherein the antigen bindingdomain binds to a stromal cell antigen.

In these an other embodiments, the antigen binding domain is an antibodyor an antigen-binding fragment thereof. In another embodiment, theantigen-binding fragment is a Fab or a scFv. In yet a furtherembodiment, the stromal cell antigen is expressed on a stromal cellpresent in a tumor microenvironment. In another embodiment, the tumor isa carcinoma. In an additional embodiment, the stromal cell antigen isfibroblast activation protein (FAP). In yet other embodiments, thecostimulatory signaling region comprises the intracellular domain of acostimulatory molecule selected from the group consisting of CD27, CD28,4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand thatspecifically binds with CD83, and any combination thereof. In anotherembodiment, the nucleic acid sequence comprises SEQ ID NO: 1 in and yetanother embodiment, the CAR is encoded by a nucleic acid sequencecomprising SEQ ID NO: 1.

The invention also includes a method for stimulating a T cell-mediatedimmune response to a stromal cell population in a mammal. The methodcomprises administering to a mammal an effective amount of a cellgenetically modified to express a CAR, wherein the CAR comprises anantigen binding domain, a transmembrane domain, a costimulatorysignaling region, and a CD3 zeta signaling domain, and wherein theantigen binding domain is selected to specifically recognize the stromalcell population.

In these and other embodiments, the antigen binding domain is anantibody or an antigen-binding fragment thereof. In another embodiment,the antigen-binding fragment is a Fab or a scFv. In yet a furtherembodiment, the stromal cell antigen is expressed on a stromal cellpresent in a tumor microenvironment. In another embodiment, the tumor isa carcinoma. In an additional embodiment, the stromal cell antigen isfibroblast activation protein (FAP). In yet other embodiments, thecostimulatory signaling region comprises the intracellular domain of acostimulatory molecule selected from the group consisting of CD27, CD28,4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand thatspecifically binds with CD83, and any combination thereof. In anotherembodiment, the nucleic acid sequence comprises SEQ ID NO: 1 in and yetanother embodiment, the CAR is encoded by a nucleic acid sequencecomprising SEQ ID NO: 1.

The invention also includes a composition comprising an anti-FAP bindingdomain. In one embodiment, the composition is an antibody, or fragmentthereof. In another embodiment, the composition is encoded by a nucleicacid sequence comprising SEQ ID NO: 3.

The invention further includes a composition comprising a cellcomprising a nucleic acid sequence encoding a chimeric antigen receptor(CAR), the CAR comprising an antigen binding domain, a transmembranedomain, a costimulatory signaling region, and a CD3 zeta signalingdomain, wherein the antigen binding domain binds to a stromal cellantigen, in combination with an antitumor vaccine.

The invention additionally includes a method of treating cancer in amammal comprising administering to the mammal a cell comprising anucleic acid sequence encoding a chimeric antigen receptor (CAR), theCAR comprising an antigen binding domain, a transmembrane domain, acostimulatory signaling region, and a CD3 zeta signaling domain, whereinthe antigen binding domain binds to a stromal cell antigen, and anantitumor vaccine.

One embodiment, the cell and the antitumor vaccine are co-administeredto the mammal. In another embodiment, the cell and the antitumor vaccineare administered to the mammal separately.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1 depicts a schematic of an exemplary CAR.

FIG. 2 is a set of graphs depicting the results of experimentsdemonstrating the specificity of mFAP-CAR T cells. The upper paneldepicts the amount of IFN-γ production of untransduced and mFAP-CARtransduced T cells alone and upon exposure to 3T3 wildtype (WT) or3T3mFAP cells. The lower panel depicts the percent killing ofuntransduced and mFAP-CAR transduced killing upon co-culture with 3T3WTor 3T3mFAP cells.

FIG. 3 is a set of graphs depicting the results of experimentsdemonstrating the expression of mFAP-CAR (right panel), compared tountransduced control (left panel).

FIG. 4 is an image depicting the presence of FAP in a mouse mesothelioma(AE17) tumor.

FIG. 5 is a graph depicting tumor size after 0, 4, and 9 days afterintravenous injection of saline (AE17), 10⁷ mouse T cells transfectedwith a retrovirus encoding GFP (MigR1) or 10⁷ mouse T cells transfectedwith mFAP-CAR (H2L).

FIG. 6 is a graph depicting the results of an experiment assessing tumorsize before and after intravenous injection of saline (WT-untreated) or10⁷ human T cells transfected with a lentivirus encoding mFAP-CAR(WT-H2L).

FIG. 7 is a graph depicting the results of an experiment assessing tumorsize after treatment with unmodified control T cells (control) orFAP-CAR T cells (FAP). T cells were injected into WT-B6 mice (upperpanel) or Knock Out Mice lacking expression of FAP (lower panel).

FIG. 8 is a schematic depicting an exemplary vector encoding a FAP-CARof the invention.

FIG. 9A depicts flow cytometric analysis of FAP expression in parental3T3 fibroblasts.

FIG. 9B depicts flow cytometric analysis of FAP expression in 3T3.FAPfibroblasts.

FIG. 9C depicts flow cytometric analysis of FAP expression in AE17.ovamouse mesothelioma cells.

FIG. 9D depicts flow cytometric analysis of FAP expression in TC1 mouselung cancer cells.

FIG. 9E depicts flow cytometric analysis of FAP expression in LKR mouselung cancer cells.

FIG. 10A depicts generation of anti-murine FAPlacZ/lacZ mABs. Mice wereimmunized with BALB/c3T3 cells transduced with lentivirus encodingfull-length muFAP.

FIG. 10B shows FACS of hybridoma supernatants screened by ELISA (notshown) and by flow cytometry of BALB/c3T3.muFAP (right peak) andBALB/c3T3.LacZ cells (left peak), respectively. The reactivity of mAb73.3 is depicted.

FIG. 10C shows lysates of BALB/c3T3.LacZ cells and BALB/c3T3.muFAP cellssubjected to IP using mAb 73.3 followed by immunoblotting with secondindependent anti-FAP mAb.

FIG. 11A depicts the structure of FAP-CAR. Total RNA of 73.3 hybridomawas extracted, reverse transcribed to cDNA, and PCR amplified andinserted into cloning vector to obtain the sequence of variable domainsof IgG heavy chain (SEQ ID NO: 8)

FIG. 11B depicts the structure of FAP-CAR and light (SEQ ID NO: 9)chains.

FIG. 11C depicts anti-muFAP CAR consisting of the anti-muFAP scFv, CD8αhinge and transmembrane domain, plus 4-1BB and CD3ζ intracellularsignaling domains, and was cloned into MigR1 retroviral vector in orderto transduce primary mouse T cells.

FIG. 11D depicts a fully mouse FAP-CAR construct, which consists of theanti-muFAP scFv, CD8α hinge and CD28 transmembrane domain, plus CD28 andCD3ζ intracellular signaling domains of mouse origin, and was clonedinto MSGV retroviral vector in order to transduce primary mouse T cells.

FIG. 12A depicts ex vivo assessment of mouse CART cells redirectedagainst FAP with retroviral transduction of mouse T cells to express GFP(MigR1) or GFP and anti-mFAP-CAR.

FIG. 12B shows the up regulation of CD69 on CAR (GFP)-positive CD8 andCD4 T cells following stimulation with BSA- or FAP-coated beads for 18hours. T cells were stimulated with anti-CD3/anti-CD28 beads as positivecontrol.

FIG. 12C shows the target-specific cytolytic activity of FAP-CAR Tcells.

FIG. 12D shows the IFNγ production of FAP-CAR T cells. VariousEffector:Target ratios of MigR1 and FAP-CAR T cells were reacted withparental 3T3 or 3T3.FAP fibroblasts for 18 hours. * Denotes statisticalsignificance between FAP-CAR-treated 3T3.FAP group versus the other 3group, p value<0.05.

FIG. 13 depicts signaling in FAP CAR T cells. FAP-CAR T cells wereexposed either BSA- or FAP-coated beads for 10 min. Cell lysates werethen prepared and immunoblotted for phospho-ERK, phospho-AKT, andphospho-IKKα/β. Anti-CD3ε antibody was used as a positive control for Tcell activation, and b-actin was immunoblotted to check for equalloading.

FIG. 14 depicts phenotypic analysis of FAP+ stromal cells in untreatedTC1 flank tumors in C57BL/6 mice. Untreated TC1 tumors were harvestedand digested with mixture of collagenases to make single cellsuspension. Cells were then stained with anti-CD45, anti-CD90 andanti-FAP. Propidium iodide was used to exclude dead cells.

FIG. 15A depicts anti-tumor activities of FAP-CAR T cells in syngeneicmice bearing TC1.

FIG. 15B depicts anti-tumor activities of FAP-CAR T cells in LKR.

FIG. 15C depicts anti-tumor activities of FAP-CAR T cells in AE17.ovatumors injected intravenously with 10 million FAP-CAR or MigR1 T cellswhen tumor reached approximately 100-150 mm³. Tumor measurements werethen followed.

FIG. 15D shows the target-specificity of FAP-CAR T cells, AE17.ova tumorcells were also injected into FAP-null C57BL/6 mice. FAP-CAR T cellswere given 7 days later. * Denotes statistical significance betweenuntreated, MigR1 and FAP-CAR-treated samples, p value<0.05.

FIG. 16A depicts anti-tumor activity of FAP-CAR T cells in CT26 and 4T1tumor models. A single dose of FAP-CAR or MigR1 T cells was injectedinto mice bearing CT26 colon cancer.

FIG. 16B depicts anti-tumor activity of FAP-CAR T cells in mammarycancer, when tumors reached approximately 100-150 mm3. Tumormeasurements were then followed. * Denotes statistical significancebetween untreated, MigR1 and FAP-CAR-treated samples, p value<0.05.

FIG. 17A depicts depletion of FAP+ cells. FAP-CAR T cells were injectedintravenously into AE17.ova tumor bearing mice when tumors reached 100mm3. At 3 days after T cell infusion, tumors were harvested and digestedto determine amount of FAP+ cell depletion.

FIG. 17B shows depletion of FAPhi cells by FAP-CAR T cells.

FIG. 17C shows depletion of FAPlo cells by FAP-CAR T cells * Denotesstatistical significance between untreated, MigR1 and FAP-CAR-treatedsamples, p value<0.05.

FIG. 18A depicts persistence and anti-tumor activities of two types ofFAP-CAR T cells employing either human 4-1BB (73.3-hBBz) or mouse CD28(73.3-m28z) co-stimulatory domain in mice. Persistence of FAP-CAR Tcells (73.3-hBBz) over time. AE17.ova-tumor bearing mice was injectedwith 10 million FAP-CAR T cells through tail vein when tumor sizereached approximately 100 mm³. Tumors were harvested 3, 7 and 10 daysafter adoptive transfer, to look for GFP+CD3+ FAP-CAR T cells (n=5). *Denotes statistical significance in lower percent CAR TILs compared tothe 3 day time point, p value<0.05.

FIG. 18B shows FAP-CAR T cells with different co-stimulatory domainpersisted similarly in vivo. Two FAP-CAR constructs, FAP-CAR-hBBz andFAP-CAR-m28z, were transduced into congnic Thy1.1 C57BL/6 mouse T cellsto determine their trafficking and persistence in tumor-bearing mice.AE17.ova tumors were injected into regular Thy1.2 C57BL/6 mice. Whentumors reached approximately 100-125 mm³, a single dose (10 million) ofThy1.1+ FAP-CAR T cells were then adoptively transferred through tailvein into mice. Tumors were harvested 3 days after adoptive transfer ofFAP-CAR T cells in mice. Tumors and pancreas were digested and made intosingle cell suspension. Cells were then stained withfluorochrome-conjugated anti-Thy1.1, together with anti-CD3 antibody todetermine percent FAP-CAR T cells in tumors and pancreas.

FIG. 18C shows in vivo anti-tumor activity of two FAP-CAR T cells.AE17.ova-tumor bearing mice was injected with 10 million FAP-CAR T cellsthrough tail vein when tumor size reached approximately 150 mm³. Tumormeasurements were then followed. * Denotes statistical significancebetween untreated and FAP-CAR-treated samples, p value<0.05.

FIG. 19A depicts in vitro activity of T cells expressing fully mouseFAPCAR. To determine target-specific cytolytic activity of two FAP-CAR Tcells, various Effector:Target ratio of MigR1 and FAP-CAR T cells werereacted with 3T3.FAP fibroblasts for 18 hours.

FIG. 19B shows IFNγ (FIG. 19B) production from FAP-CAR T cells reactedwith 3T3.FAP at effector:target ratio of 10:1 for 18 hours. *Denotesstatistical significance between MigR1 versus FAP-CAR-treated groups, pvalue<0.05.

FIG. 19C shows and TNF production from FAP-CAR T cells.

FIG. 20 is a Table (Table 1) depicting depletion of FAP⁺ cells in flanktumors post FAP-CAR treatment.

FIG. 21A depicts enhanced therapeutic response of FAP-CAR T cells bygiving two doses of FAP-CAR T cells.

FIG. 21B shows the deletion of a negative T cell regulator DGKZ.

FIG. 21C shows the combination of another immunotherapy. Mice withAE17.ova flank tumors were injected intravenously with FAP-CAR T cellswhen tumors were approximately 100 mm³. The overall efficacy of FAP-CART cells was enhanced when a second dose of FAP-CAR T cells was given aweek after the first dose. Grey arrow indicates the injection time ofthe second dose of FAP-CAR T cells. Alternatively, efficacy of FAP-CAR Tcells could be enhanced when a negative intracellular regulator DGKZ wasdeleted in those T cells. * Denotes statistical significance betweenuntreated and FAP-CAR-treated samples, p value<0.05. # Denotesstatistical significance between single dose FAP-CAR treated groupversus double dose group or DGKZ KO FAP-CAR treated group. FIG. 21Cshows FAP-CAR T cells enhance efficacy of cancer vaccine. TC1 tumorcells were inoculated into the right flanks of C57BL/6 mice. When tumorsreached 200 mm, one dose of Ad.E7 (10⁹ pfu) was given to the micecontralaterally to their flank tumors (black arrow). FAP-CAR T cells (10million cells) were given 4 days later (gray arrow). Tumor measurementswere then followed. The values are expressed as the mean±SEM (n=5). *Denotes significant difference between untreated and the combo groups(p<0.05).

FIG. 22A depicts deletion of DGKζ enhanced cytotoxicity of FAPCAR Tcells. Splenic T cells were isolated from intact C57BL/6 mice, as wellas DGKζ knockout mice. Isolated T cells were then activated, transducedwith FAP-CAR and expanded. A week later, FAP-CAR T cells with or withoutDGKζ deletion were reacted with 3T3 or 3T3.FAP fibroblasts for 18 hoursto determine cytotoxicity. * Denotes statistical significance betweenuntreated and two FAP-CARtreated samples, p value<0.05. # Denotesstatistical significance between WT and DGKζ KO FAP-CAR-treated samples,p value<0.05.

FIG. 22B depicts deletion of DGKζ enhanced IFNγ production of FAPCAR Tcells. Splenic T cells were isolated from intact C57BL/6 mice, as wellas DGKζ knockout mice. Isolated T cells were then activated, transducedwith FAP-CAR and expanded. A week later, FAP-CAR T cells with or withoutDGKζ deletion were reacted with 3T3 or 3T3.FAP fibroblasts for 18 hoursto determine IFNγ production. * Denotes statistical significance betweenuntreated and two FAP-CARtreated samples, p value<0.05. # Denotesstatistical significance between WT and DGKζ KO FAP-CAR-treated samples,p value<0.05.

FIG. 23 depicts that DGKζ knockout FAP-T cells persisted longer thanwildtype FAP-CAR T cells. AE17.ova tumor mice were adoptivelytransferred with 10 million wild-type or DGKζ knockout FAP-CAR T cellswhen tumors reached 100 mm3. Tumors were harvested 11 dayspost-injection to determine persistence of T cells. Percent FAP-CAR Tcells were determined using flow cytometry. * Denotes statisticalsignificance between WT and DGKζ KO FAP-CAR-treated samples, pvalue<0.05.

FIG. 24A depicts adaptive immune response plays a key role in FAP-CAR Tcells-induced antitumor response. AE17.ova tumors were injected intoboth C57BL/6.

FIG. 24B shows AE17.ova tumors injected into NSG mice. When tumorsreached approximately 100-125 mm³, a single dose (10 million) of FAP-CART cells were then adoptively transferred through tail vein into mice.Tumor measurements were then followed. * Denotes statisticalsignificance between untreated and FAP-CAR-treated samples, pvalue<0.05. FAP-CAR induced infiltration of antigen-specific CD8 T cellsinto tumors.

FIG. 24C shows TC1 tumors harvested 8 days after adoptive transfer ofFAP-CAR T cells in mice.

FIG. 24D shows AE17.ova tumors were harvested 8 days after adoptivetransfer of FAP-CAR T cells in mice. Tumors were digested and made intosingle cell suspension. Cells were then stained withfluorochrome-conjugated tetramer loaded with E7- orSIINFEKEL(ova)-peptide, together with anti-CD8 antibody to determinepercent tumor-specific CD8 T cells in tumors. * Denotes statisticalsignificance between untreated, FAP-CAR-treated samples, p value<0.05.significance between WT and DGKζ KO FAP-CAR-treated samples, pvalue<0.05.

FIG. 24E shows TC1 tumors harvested 8 days after adoptive transfer ofMigR1 T cells in mice.

FIG. 24F shows AE17.ova tumors were harvested 8 days after adoptivetransfer of FAP-CAR T cells in mice. Tumors were digested and made intosingle cell suspension. Cells were then stained withfluorochrome-conjugated tetramer loaded with E7- orSIINFEKEL(ova)-peptide, together with anti-CD8 antibody to determinepercent tumor-specific CD8 T cells in tumors. * Denotes statisticalsignificance between untreated, MigR1-treated samples, p value<0.05.significance between WT and DGKζ KO FAP-CAR-treated samples, pvalue<0.05.

FIG. 25A depicts infiltration of endogenous TILs following treatment ofFAP-CAR T cells. AE17.ova tumor bearing mice were given FAP-CAR or MigR1T cells when tumors reached approximately 100 mm3. Tumors were harvested3 days later to determine percent T cells infiltrating AE17.ova tumorsfollowing treatment of FAP-CAR T cells.

FIG. 25B depicts infiltration of endogenous TILs following treatment ofFAP-CAR T cells harvested 8 days later to determine percent T cellsinfiltrating AE17.ova tumors following treatment of FAP-CAR T cells. *Denotes statistical significance between untreated, MigR1 andFAP-CAR-treated groups, p value<0.05.

FIG. 26A depicts FAP-CAR T cells activate endogenous T cells. AE17.ovatumors were injected intravenously with 10 million FAP-CAR or MigR1 Tcells when tumor reached approximately 100 mm³. At 3 and 8 daysfollowing adoptive transfer, tumors were harvested and digested to checkfor number of TNF producing cells.

FIG. 26B shows IFNγ-producing cells.

FIG. 26C shows the number of 4-1BB+ TILs.

FIG. 26D shows the number of CD69+ TILs. * Denotes statisticalsignificance between untreated, MigR1 and FAP-CAR-treated samples, pvalue<0.05.

FIGS. 27A-27F show the body weights of FAP-CAR T cell-treated miceremained unchanged or increased. Body weight of tumor bearing micereceiving a single dose of FAP-CAR T cells were monitored over time.

FIG. 28A depicts that bone marrows showed normal histology followingtreatment with FAP-CAR T cells. Representative H&E sections of femurbones are shown from control untreated tumor-bearing mice.

FIG. 28B shows representative H&E sections of femur bones fromtumor-bearing mice treated with MigR1 T cells.

FIG. 28C shows representative H&E sections of femur bones fromtumor-bearing mice treated with FAP-CAR T cells. Femurs were harvestedone week after T cell infusion.

FIG. 29A depicts pancreas histology. Representative H&E sections of thepancreas are shown from control untreated tumor-bearing mice.

FIG. 29B shows representative H&E sections of the pancreas fromtumor-bearing mice treated with wild-type FAP-CAR T cells. No changeswere seen in the organs treated with WT FAP-CAR T cells.

FIG. 29C shows representative H&E sections of the pancreas fromtumor-bearing mice treated with DGK knockout FAP-CAR T cells. Organswere harvested one week after T cell infusion. Pancreatic Islets ofLangerhans are marked by an asterisk. Blood vessels are labeled with a“V”. In mice treated with DGK knockout FAPCAR T cells, however, focalareas of lymphocytes were noted in a peri-islet (white arrows) andperi-vascular (white arrowheads) location.

FIG. 30A depicts activation-induced cell death of FAP-CAR T cells.Comparison of in vivo persistence between MigR1 and FAP-CAR T cells.Tumors were harvested 3 and 8 days following adoptive transfer of 10million FAP-CAR T cells in AE17.ova tumor bearing mice. * Denotesstatistical significance in lower percent TILs compared to the 3 daytime point, p value<0.05. # Denotes statistical significance in lowerpercent FAP-CAR TILs compared to MigR1 TILs at the same time point, pvalue<0.05.

FIG. 30B shows that the FAP-CAR T cells died soon after antigenstimulation. MigR1 and FAP-CAR T cells with either human or mouseintracellular domains were exposed to either BSA- or FAP-coated beads.Amount of live cells were counted every day for 3 days, by trypan bluestaining.

FIGS. 31A-31D depict differential FAP expression on tumor and pancreaticFAP+ stromal cells. Tumors and pancreas from different tumor bearingmice were harvested and digested to form single cell suspension. Cellswere then stained with biotin-conjugated anti-FAP antibody andstreptavidin-conjugated fluorochrome, together with anti-CD90 andanti-CD45 antibodies. The mean fluorescence intensity of FAP expressionon CD90+CD45-cells in tumors and pancreas were compared.

FIG. 32 is a graph depicting in vivo efficacy of FAP-CAR T cells inmouse pancreatic cancer model.

FIG. 33 is a graph depicting in vivo efficacy of FAP-CAR T cells in anautochthonous lung cancer model.

FIG. 34 is a graph depicting CAR expression in human T cells afterelectroporation with anti-mouse FAP CAR mRNA.

FIG. 35 is a graph depicting killing of FAP-expressing cells bymRNA-transfected T cells.

FIG. 36 is a graph depicting mRNA CAR T cells in A549 xenograft model.

FIG. 37 is a graph depicting cytolytic activity of anti-huFAP CAR Tcells against 3T3 expressing human FAP.

FIG. 38 is a graph depicting cytolytic activity of anti-huFAP CAR Tcells against FAP-null 3T3 cells.

FIG. 39 is a graph depicting IFNg production of anti-huFAP CAR T cellsagainst 3T3 expressing human FAP.

FIG. 40 is a graph depicting IFNg of anti-huFAP CAR T cells againstFAP-null 3T3 cells.

FIG. 41 is series of images and a graph depicting that administration ofFAP-CAR T cells did not interfere with host wound healing response.

FIG. 42 is a series of graphs depicting that administration of FAP-CAR Tcells did not cause weight loss or toxicity in pancreas.

FIG. 43 is a graph depicting decrease in FAP-positive lung stromal cellsfollowing treatment of FAPCAR T cells in mice with pulmonary fibrosis.

DETAILED DESCRIPTION

The invention relates to compositions and methods for targeting stromalcells in the treatment of cancer. Immunotherapy for cancer, whetheradoptive T cell therapy, antibody- or vaccine-based, has to date beenfocused primarily on targeting antigens expressed by the neoplasticcells. It is now evident that other components including stromal cells,infiltrating inflammatory/immune cells, vasculature and extracellularmatrix that comprise the tumor microenvironment, are also required foror promote tumor growth and metastasis and therefore present additionaltherapeutic targets.

In one embodiment, the present invention comprises compositions thattarget fibroblast activation protein (FAP). FAP is a cell surfaceprotease that is expressed on the vast majority of stromal cells invirtually all human carcinomas. In one embodiment, the present inventionprovides an antibody that specifically binds to FAP. In one embodiment,the present invention provides compositions comprising an anti-FAPantibody, or an FAP binding fragment thereof. Non-limiting Examples ofcompositions targeting FAP encompassed by the present invention includeantibodies, immunoconjugates, antibody conjugates, vaccines, andchimeric antigen receptors (CARs) that target FAP.

The present invention has certain advantages over prior art cancertreatments in that antibody conjugates can have limited tumorpenetration and often induce an immune response in the host, andvaccination may lead to long lasting endogenous immunity to FAP. Thepresent compositions, i.e., using an anti-FAP CAR T cell is designed tocircumvent these limitations.

In one embodiment, the present invention comprises a method to treatcancer comprising administering to a subject a composition targeting astromal cell in a tumor microenvironment. In one embodiment, thecomposition comprises an anti-FAP antibody, or a FAP binding fragmentthereof. In one embodiment, administration of the composition reducesand/or eliminates the stromal cell population. In one embodiment,administration of the composition reduces and/or eliminates the cancer.

In one embodiment, the present invention provides a CAR comprising ananti-FAP antibody, or an FAP binding fragment thereof. CARs aremolecules that combine antibody-based specificity for a desired antigenwith a T cell receptor-activating intracellular domain to generate achimeric protein that exhibits a specific anti-antigen cellular immuneactivity. In some embodiments, a CAR targeting FAP is preferred. A CARtargeting FAP is referred to herein as an FAP-CAR.

The present invention provides for the incorporation of an anti-FAPbinding domain into a CAR in order to treat and eliminate tumors. Asdiscussed elsewhere herein, FAP-CAR displays specificity in recognizingFAP expressing cells. Further, T cells transduced with FAP-CAR are ableto reduce tumor size.

The present invention provides for the targeting of stromal cells in thetreatment of cancer, whether or not the stromal cells themselves arecancer cells. A potential limitation of CARs targeted to tumor cells isthat antigen expression on tumors is heterogeneous and subject toantigen loss. Therefore, in some embodiments, the present invention isdirected towards the targeting of the more genetically stable stromalcells and thereby avoids the problems associated with targeting tumorantigens directly. In some embodiments, the more genetically stablestromal cells are not a cancer cell.

The present invention relates generally to the use of T cellsgenetically modified to stably express a desired CAR. T cells expressinga CAR are referred to herein as CAR T cells or CAR modified T cells.Preferably, the cell can be genetically modified to stably express anantibody binding domain (e.g., anti-FAP) on its surface, conferringnovel antigen specificity that is MHC independent. In some instances,the T cell is genetically modified to stably express a CAR that combinesan antigen recognition domain of a specific antibody with anintracellular domain of the CD3-zeta chain or FcγRI protein into asingle chimeric protein.

In one embodiment, the CAR of the invention comprises an extracellulardomain having an antigen recognition domain, a transmembrane domain, anda cytoplasmic domain. In one embodiment, the transmembrane domain thatnaturally is associated with one of the domains in the CAR is used. Inanother embodiment, the transmembrane domain can be selected or modifiedby amino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex. Insome embodiments, the extracellular domain also comprises a hingedomain.

With respect to the cytoplasmic domain, the CAR of the invention can bedesigned to comprise a signaling domain of a costimulatory molecule. Forexample, the CAR of the invention can be designed to comprise the CD28and/or 4-1BB signaling domain by itself or be combined with any otherdesired cytoplasmic domain(s) useful in the context of the CAR of theinvention. In one embodiment, the cytoplasmic domain of the CAR can bedesigned to further comprise the signaling domain of CD3-zeta. Forexample, the cytoplasmic domain of the CAR can include, but is notlimited to, CD3-zeta, 4-1BB and CD28 signaling modules, and combinationsthereof (FIG. 1). Accordingly, the invention provides CAR T cells andmethods of their use for adoptive therapy.

In one embodiment, the CAR T cells of the invention can be generated byintroducing a lentiviral vector comprising the desired CAR, for examplea CAR comprising anti-FAP, a transmembrane domain, and CD3zeta signalingdomains, into the cells. In one embodiment, the CAR T cells of theinvention are able to replicate in vivo resulting in long-termpersistence that can lead to sustained tumor control.

In another embodiment, the CAR T cells of the invention can be generatedby transfecting an RNA encoding the desired CAR, for example a CARcomprising anti-FAP, a transmembrane domain, and CD3-zeta signalingdomains, into the cells. In one embodiment, the CAR is transientlyexpressed in the genetically modified CAR T cells.

In one embodiment the invention relates to administering a geneticallymodified T cell expressing a CAR for the treatment of a patient havingcancer or at risk of having cancer using lymphocyte infusion.Preferably, autologous lymphocyte infusion is used in the treatment.Autologous PBMCs are collected from a patient in need of treatment and Tcells are activated and expanded using the methods described herein andknown in the art and then infused back into the patient.

In one embodiment, the invention relates to genetically modified T cellsexpressing a CAR for the treatment of a patient with cancer. The presentinvention is based upon the finding that the inclusion of the anti-FAPantigen binding domain in a CAR allows specific recognition of FAPexpressing cells and tumor reduction.

In yet another embodiment, the invention relates generally to thetreatment of a patient at risk of developing cancer. The invention alsoincludes treating a malignancy or an autoimmune disease in whichchemotherapy and/or immunotherapy in a patient results in significantimmunosuppression in the patient, thereby increasing the risk of thepatient of developing cancer.

The invention includes using T cells expressing an FAP-CAR, includingthe CD3-zeta intracellular domain (also referred to as FAP-CAR T cells).In one embodiment, the FAP-CAR T cells of the invention can undergorobust in vivo T cell expansion and can establish FAP-specific memorycells that persist at high levels for an extended amount of time inblood and bone marrow. In some instances, the FAP-CAR T cells of theinvention infused into a patient can eliminate tumor cells in vivo inpatients with cancer. The invention includes any anti-FAP binding domainfused with one or more intracellular domains selected from the group ofa CD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD3zetasignal domain, and any combination thereof.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or in some instances ±10%, or in some instances ±5%,in some instances ±1%, and in some instances ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

“Activation”, as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

As used herein, the term “Chimeric Antigen Receptor” or alternatively a“CAR” refers to a recombinant polypeptide construct comprising at leastan extracellular antigen binding domain, a transmembrane domain and acytoplasmic signaling domain comprising a functional signaling domainderived from a stimulatory molecule as defined below. In one aspect, thestimulatory molecule is the zeta chain associated with the T cellreceptor complex. In one aspect, the cytoplasmic signaling domainfurther comprises one or more functional signaling domains derived fromat least one costimulatory molecule as defined below. In one aspect, thecostimulatory molecule is chosen from 4 1BB (i.e., CD137) and/or CD28.In one aspect, the CAR comprises a chimeric fusion protein comprising anextracellular antigen recognition domain, a transmembrane domain and anintracellular signaling domain comprising a functional signaling domainderived from a stimulatory molecule. In one aspect, the CAR comprises achimeric fusion protein comprising an extracellular antigen recognitiondomain, a transmembrane domain and an intracellular signaling domaincomprising a functional signaling domain derived from a co-stimulatorymolecule and a functional signaling domain derived from a stimulatorymolecule. In one aspect, the CAR comprises a chimeric fusion proteincomprising an extracellular antigen recognition domain, a transmembranedomain and an intracellular signaling domain comprising two functionalsignaling domains derived from one or more co-stimulatory molecule(s)and a functional signaling domain derived from a stimulatory molecule.In one aspect, the CAR comprises a chimeric fusion protein comprising anextracellular antigen recognition domain, a transmembrane domain and anintracellular signaling domain comprising at least two functionalsignaling domains derived from one or more co-stimulatory molecule(s)and a functional signaling domain derived from a stimulatory molecule.In one aspect the CAR comprises an optional leader sequence at theamino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CARfurther comprises a leader sequence at the N-terminus of theextracellular antigen recognition domain, wherein the leader sequence isoptionally cleaved from the scFv domain during cellular processing andlocalization of the CAR to the cellular membrane.

As used herein, a “signaling domain” is the functional portion of aprotein which acts by transmitting information within the cell toregulate cellular activity via defined signaling pathways by generatingsecond messengers or functioning as effectors by responding to suchmessengers.

As used herein, “a stromal cell antigen” refers to an antigen expressedon or by a stromal cell.

As used herein, ‘FAP” refers to fibroblast activation protein. The termshould be construed to include not only fibroblast activation protein,but variants, homologs, fragments and portions thereof to the extentthat such variants, homologs, fragments and portions thereof retain theactivity of FAP as disclosed herein.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can bepolyconal or monoclonal, multiple or single chain, or intactimmunoglobulins, and may be derived from natural sources or fromrecombinant sources. Antibodies such as IgG are typically tetramers ofimmunoglobulin molecules.

The term “antibody fragment” refers to at least one portion of an intactantibody, or recombinant variants thereof, and refers to the antigenbinding domain, e.g., an antigenic determining variable region of anintact antibody, that is sufficient to confer recognition and specificbinding of the antibody fragment to a target, such as an antigen.Examples of antibody fragments include, but are not limited to, Fab,Fab′, F(ab′)₂, and Fv fragments, scFv antibody fragments, linearantibodies, single domain antibodies such as sdAb (either VL or VH),camelid VHH domains, and multi-specific antibodies formed from antibodyfragments. The term “scFv” refers to a fusion protein comprising atleast one antibody fragment comprising a variable region of a lightchain and at least one antibody fragment comprising a variable region ofa heavy chain, wherein the light and heavy chain variable regions arecontiguously linked via a short flexible polypeptide linker, and capableof being expressed as a single chain polypeptide, and wherein the scFvretains the specificity of the intact antibody from which it is derived.Unless specified, as used herein an scFv may have the VL and VH variableregions in either order, e.g., with respect to the N-terminal andC-terminal ends of the polypeptide, the scFv may comprise VL-linker-VHor may comprise VH-linker-VL.

The portion of the CAR composition of the invention comprising anantibody or antibody fragment thereof may exist in a variety of formswhere the antigen binding domain is expressed as part of a contiguouspolypeptide chain including, for example, a single domain antibodyfragment (sdAb), a single chain antibody (scFv) and a humanized antibody(Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies:A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988,Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science242:423-426). In one aspect, the antigen binding domain of a CARcomposition of the invention comprises an antibody fragment. In afurther aspect, the CAR comprises an antibody fragment that comprises ascFv.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in antibody molecules in theirnaturally occurring conformations, and which normally determines theclass to which the antibody belongs

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in antibody molecules in theirnaturally occurring conformations. Kappa (κ) and lambda (λ) light chainsrefer to the two major antibody light chain isotypes.

By the term “recombinant antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage or yeast expressionsystem. The term should also be construed to mean an antibody which hasbeen generated by the synthesis of a DNA molecule encoding the antibodyand which DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using recombinant DNA or amino acid sequencetechnology which is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatcan provoke an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to encodepolypeptides that elicit the desired immune response. Moreover, askilled artisan will understand that an antigen need not be encoded by a“gene” at all. It is readily apparent that an antigen can be generatedsynthesized or can be derived from a biological sample, or can bemacromolecules besides a polypeptide. Such a biological sample caninclude, but is not limited to a tissue sample, a tumor sample, a cellor fluid with other biological components.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect which can be manifested by various means, including but notlimited to, a decrease in tumor volume, a decrease in the number oftumor cells, a decrease in the number of metastases, an increase in lifeexpectancy, a decrease in tumor cell proliferation, a decrease in tumorcell survival, or amelioration of various physiological symptomsassociated with the cancerous condition. An “anti-tumor effect” can alsobe manifested by the ability of the peptides, polynucleotides, cells andantibodies of the invention in prevention of the occurrence of tumor inthe first place.

The term “auto-antigen” means any self-antigen which is recognized bythe immune system as being foreign. Auto-antigens comprise, but are notlimited to, cellular proteins, phosphoproteins, cellular surfaceproteins, cellular lipids, nucleic acids, glycoproteins, including cellsurface receptors.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addision's disease, alopecia greata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to whom it is later to be re-introducedinto the individual.

“Allogeneic” refers to any material derived from a different animal ofthe same species as the individual to whom the material is introduced.Two or more individuals are said to be allogeneic to one another whenthe genes at one or more loci are not identical. In some aspects,allogeneic material from individuals of the same species may besufficiently unlike genetically to interact antigenically.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody or antibodyfragment containing the amino acid sequence. Such conservativemodifications include amino acid substitutions, additions and deletions.Modifications can be introduced into an antibody or antibody fragment ofthe invention by standard techniques known in the art, such assite-directed mutagenesis and PCR-mediated mutagenesis. Conservativeamino acid substitutions are ones in which the amino acid residue isreplaced with an amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one ormore amino acid residues within the CDR regions of an antibody orantibody fragment of the invention can be replaced with other amino acidresidues from the same side chain family and the altered antibody orantibody fragment can be tested for the ability to bind CD19 using thefunctional assays described herein.

By the term “stimulation,” used in the context of CART, is meant aprimary response induced by binding of a stimulatory molecule (e.g., aTCR/CD3 complex) with its cognate ligand thereby mediating a signaltransduction event, such as, but not limited to, signal transduction viathe TCR/CD3 complex. Stimulation can mediate altered expression ofcertain molecules, such as downregulation of TGF-β, and/orreorganization of cytoskeletal structures, and the like.

A “stimulatory molecule,” used in the context of CART, means a moleculeexpressed by a T cell that provide the primary cytoplasmic signalingsequence(s) that regulate primary activation of the TCR complex in astimulatory way for at least some aspect of the T cell signalingpathway. In one aspect, the primary signal is initiated by, forinstance, binding of a TCR/CD3 complex with an MEW molecule loaded withpeptide, and which leads to mediation of a T cell response, including,but not limited to, proliferation, activation, differentiation, and thelike. Primary cytoplasmic signaling sequences that act in a stimulatorymanner may contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs. Examples of ITAM containingprimary cytoplasmic signaling sequences that are of particular use inthe invention include those derived from TCR zeta, FcR gamma, FcR beta,CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (alsoknown as “ICOS”) and CD66d. In a specific CAR of the invention, thecytoplasmic signaling molecule in any one or more CARS of the invention,including CARs comprises a cytoplasmic signaling sequence derived fromCD3-zeta. In a specific CAR of the invention, the cytoplasmic signalingsequence derived from CD3-zeta is the sequence provided as SEQ ID NO:7or the equivalent residues from a non-human species, e.g., mouse,rodent, monkey, ape and the like.

An “antigen presenting cell,” as used herein, means an immune systemcell such as an accessory cell (e.g., a B-cell, a dendritic cell, andthe like) that displays foreign antigens complexed with majorhistocompatibility complexes (MHC's) on their surfaces. T-cells mayrecognize these complexes using their T-cell receptors (TCRs). APCsprocess antigens and present them to T-cells.

As used herein “zeta” or alternatively “zeta chain”, “CD3-zeta” or“TCR-zeta” is defined as the protein provided as GenBan accno.BAG36664.1, or the equivalent residues from a non-human species, e.g.,mouse, rodent, monkey, ape and the like, and a “zeta stimulatory domain”or alternatively a “CD3-zeta stimulatory domain” or a “TCR-zetastimulatory domain” is defined as the amino acid residues from thecytoplasmic domain of the zeta chain that are sufficient to functionallytransmit an initial signal necessary for T cell activation. In oneaspect the cytoplasmic domain of zeta comprises residues 52 through 164of GenBank accno. BAG36664.1 or the equivalent residues from a non-humanspecies, e.g., mouse, rodent, monkey, ape and the like, that arefunctional orthologs thereof. In one aspect, the “zeta stimulatorydomain” or a “CD3-zeta stimulatory domain” is the sequence provided asSEQ ID NO7.

A “costimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a costimulatory ligand, therebymediating a costimulatory response by the T cell, such as, but notlimited to, proliferation. Costimulatory molecules are cell surfacemolecules other than antigen receptors or their ligands that arerequired for an efficient immune response. Costimulatory moleculesinclude, but are not limited to an MEW class I molecule, BTLA and a Tollligand receptor, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1(CD11a/CD18) and 4-1BB (CD137).

As used herein “4-1BB” is defined as member of the TNFR superfamily withan amino acid sequence provided as GenBank accno. AAA62478.2, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like; and a “4-1BB costimulatory domain” are definedamino acid residues 214-255 of GenBank accno. AAA62478.2, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like. In one aspect, the “4-1BB costimulatorydomain” is the sequence provided as SEQ ID NO:6 or the equivalentresidues from a non-human species, e.g., mouse, rodent, monkey, ape andthe like.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or a RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

A “transfer vector” is a composition of matter which comprises anisolated nucleic acid and which can be used to deliver the isolatednucleic acid to the interior of a cell. Numerous vectors are known inthe art including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, and viruses. Thus, the term “transfer vector” includes anautonomously replicating plasmid or a virus. The term should also beconstrued to further include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into cells, such as, for example,polylysine compounds, liposomes, and the like. Examples of viraltransfer vectors include, but are not limited to, adenoviral vectors,adeno-associated virus vectors, retroviral vectors, lentiviral vectors,and the like.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

A “lentiviral vector” is a vector derived from at least a portion of alentivirus genome, including especially a self-inactivating lentiviralvector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009).Other Examples or lentivirus vectors that may be used in the clinic asan alternative to the pELPS vector, include but not limited to, e.g.,the LENTIVECTOR® gene delivery technology from Oxford BioMedica, theLENTIMAX™ vector system from Lentigen and the like. Nonclinical types oflentiviral vectors are also available and would be known to one skilledin the art.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared X 100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the nucleicacid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994))

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

The term “overexpressed” antigen or “overexpression” of the antigen isintended to indicate an abnormal level of expression of the antigen in acell from a disease area like a solid tumor within a specific tissue ororgan of the patient relative to the level of expression in a normalcell from that tissue or organ. Patients having solid tumors or ahematological malignancy characterized by overexpression of the antigencan be determined by standard assays known in the art.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type corresponding to the promoter.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody or antibody fragment which recognizes andbinds with a specific antigen, but does not substantially recognize orbind other molecules in a sample. For example, an antibody thatspecifically binds to an antigen from one species may also bind to thatantigen from one or more species. But, such cross-species reactivitydoes not itself alter the classification of an antibody as specific. Inanother example, an antibody that specifically binds to an antigen mayalso bind to different allelic forms of the antigen. However, such crossreactivity does not itself alter the classification of an antibody asspecific. In some instances, the terms “specific binding” or“specifically binding,” can be used in reference to the interaction ofan antibody, a protein, or a peptide with a second chemical species, tomean that the interaction is dependent upon the presence of a particularstructure (e.g., an antigenic determinant or epitope) on the chemicalspecies; for example, an antibody recognizes and binds to a specificprotein structure rather than to proteins generally. If an antibody isspecific for epitope “A”, the presence of a molecule containing epitopeA (or free, unlabeled A), in a reaction containing labeled “A” and theantibody, will reduce the amount of labeled A bound to the antibody.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The present invention provides compositions and methods for treatingcancer. The cancer may be a hematological malignancy, a solid tumor, aprimary or a metastasizing tumor.

In one embodiment, the invention provides a composition targeting thestromal cell population in a tumor microenvironment. The advantages oftargeting stromal cells are: i) that compared to neoplastic cells, therate of mutation in stromal cells is dramatically lower and thereforestromal cells are less prone to developing resistance to therapy; ii)the tumor promoting effects of stromal cells are common to multipletumor types and therefore the same stromal cell targeted therapies canbe indicated in multiple tumor types; iii) combining therapies directedagainst neoplastic cells with therapies directed against stromal cellscan have synergistic tumoricidal activity; iv) pro-tumorigenic changesin stromal cells occur and in at least some cases are required toestablish the so-called pre-metastatic niche. Therefore, in variousembodiments, targeting stromal cells markedly reduces the development ofdistal metastases which are the cause of the vast majority ofcancer-related deaths. The effective targeting of stromal cells requiresa stromal cell specific antigen. The stromal cell antigen to targetusing a targeted therapy should be one expressed on the cell surface ofa majority of stromal cells in desmoplastic tumors in which stromalcells support tumor initiation, progression and metastasis.

In one embodiment, the present invention provides a compositioncomprising a FAP binding domain. In one embodiment, the compositioncomprises an anti-FAP antibody, or a FAP binding fragment thereof.Non-limiting examples of compositions which comprise a FAP bindingdomain include an antibody, an immunoconjugate, an antibody conjugate,and a chimeric antigen receptor (CAR). The present invention is partlybased on the generation of an anti-FAP antibody.

In one embodiment, the invention provides a cell (e.g., T cell)engineered to express a CAR comprising an FAP binding domain, whereinthe CAR T cell exhibits an antitumor property. The CAR of the inventioncan be engineered to comprise an extracellular domain having an FAPbinding domain fused to an intracellular signaling domain of the T cellantigen receptor complex zeta chain (e.g., CD3-zeta). The CAR of theinvention when expressed in a T cell is able to redirect antigenrecognition based on the FAP antigen binding specificity.

The present invention provides targeting to stromal cells, rather thantumor cells directly, as it was seen that stromal cells existing in thetumor microenvironment have tumorigenic activity. For example, stromalcells in tumor microenvironments promote tumor growth and metastasis.Therefore, targeting of FAP expressing stromal cells affects a tumorcell so that the tumor cell fails to grow, is prompted to die, orotherwise is affected so that the tumor burden in a patient isdiminished or eliminated. The FAP binding domain is preferably fusedwith an intracellular domain from one or more of a costimulatorymolecule and a zeta chain. Preferably, the antigen binding domain isfused with one or more intracellular domains selected from the group ofa CD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD3zetasignal domain, and any combination thereof.

In some embodiments, the present invention is directed to a retroviralor lentiviral vector encoding a CAR that is stably integrated into a Tcell and stably expressed therein. In other embodiments, the presentinvention is directed to an RNA encoding CAR that is transfected into aT cell and transiently expressed therein. Transient, non-integratingexpression of CAR in a cell mitigates concerns associated with permanentand integrated expression of CAR in a cell.

Anti-FAP Antibodies

The present invention provides an anti-FAP antibody and compositionscomprising an anti-FAP antibody, or fragment thereof. The compositionsof the invention are used to target FAP-expressing stromal cells presentin the tumor microenvironment, in the treatment of cancer.

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (s.c.) or intraperitoneal (i.p.) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobertzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succunic anhydride, SOCl₂.

Monoclonal anti-FAP antibodies of the invention can be generated using awide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof. Antibodies are highly specific, being directed against a singleantigenic site. Furthermore, in contrast to conventional (polyclonal)antibody preparations which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the FAP antigen.For example, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler et al., Nature, 256:495 (1975), which can be used to generatemurine antibodies (or antibodies derived from other nonhuman mammals,e.g., rat, goat, sheep, cows, camels, etc.), or human antibodies derivedfrom transgenic animals (see, U.S. Pat. Nos. 6,075,181, 6,114,598,6,150,584 and 6,657,103). Alternatively, the monoclonal antibodies canbe made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567)and include chimeric and humanized antibodies. The “monoclonalantibodies” may also be isolated from phage antibody libraries using thetechniques described in Clackson et al., Nature, 352:624-628 (1991) andMarks et al., J. Mol. Biol., 222:581-597 (1991), for example.

An engineered anti-FAP antibody can be produced by any means known inthe art, including, but not limited to those techniques described hereinand improvements to those techniques. Large-scale high-yield productiontypically involves culturing a host cell that produces the engineeredanti-FAP antibody and recovering the anti-FAP antibody from the hostcell culture.

Monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in Monoclonal Antibodies andT-Cell Hybridomas, 563-681 (Elsevier, N.Y., 1981) (said referencesincorporated by reference in their entireties). For example, in thehybridoma method, a mouse or other appropriate host animal, such as ahamster or macaque monkey, is immunized to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the protein used for immunization. Alternatively, lymphocytesmay be immunized in vitro. Lymphocytes then are fused with myeloma cellsusing a suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice,pp. 59-103 (Academic Press, 1986)).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RFMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the anti-FAP antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the anti-FAP antibodies). Thehybridoma cells serve as a preferred source of such DNA. Once isolated,the DNA may be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofanti-FAP antibodies in the recombinant host cells.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. In particular, DNA sequences encoding V_(H) and V_(L)domains are amplified from animal cDNA libraries (e.g., human or murinecDNA libraries of affected tissues). The DNA encoding the V_(H) andV_(L) domains are recombined together with an scFv linker by PCR andcloned into a phagemid vector. The vector is electroporated in E. coliand the E. coli is infected with helper phage. Phage used in thesemethods are typically filamentous phage including fd and M13 and theV_(H) and V_(L) domains are usually recombinantly fused to either thephage gene III or gene VIII. Phage expressing an antigen binding domainthat binds to a particular antigen can be selected or identified withantigen, e.g., using labeled antigen or antigen bound or captured to asolid surface or bead. Examples of phage display methods that can beused to make the antibodies of the present invention include thosedisclosed in Brinkman et al., 1995, J. Immunol. Methods, 182:41-50; Ameset al., 1995, J. Immunol. Methods, 184:177-186; Kettleborough et al.,1994, Eur. J. Immunol., 24:952-958; Persic et al., 1997, Gene, 187:9-18;Burton et al., 1994, Advances in Immunology, 57:191-280; InternationalApplication No. PCT/GB91/O1 134; International Publication Nos. WO90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO95/15982, WO 95/20401, and WO97/13844; and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and5,969,108; each of which is incorporated herein by reference in itsentirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceFab, Fab′ and F(ab′)₂ fragments can also be employed using methods knownin the art such as those disclosed in PCT Publication No. WO 92/22324;Mullinax et al., 1992, BioTechniques, 12(6):864-869; Sawai et al., 1995,AJRI 34:26-34; and Better et al., 1988, Science, 240:1041-1043 (saidreferences incorporated by reference in their entireties).

In a further embodiment, antibodies may be isolated from antibody phagelibraries generated using the techniques described in McCafferty et al.,Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991).Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolationof murine and human antibodies, respectively, using phage libraries.Chain shuffling can be used in the production of high affinity (nMrange) human antibodies (Marks et al., Bio/Technology, 10:779-783(1992)), as well as combinatorial infection and in vivo recombination asa strategy for constructing very large phage libraries (Waterhouse etal., Nuc. Acids. Res., 21:2265-2266 (1993)). Thus, these techniques areviable alternatives to traditional monoclonal antibody hybridomatechniques for isolation of anti-FAP antibodies.

To generate whole antibodies, PCR primers including V_(H) or V_(L)nucleotide sequences, a restriction site, and a flanking sequence toprotect the restriction site can be used to amplify the V_(H) or V_(L)sequences in scFv clones.

Utilizing cloning techniques known to those of skill in the art, the PCRamplified V_(H) domains can be cloned into vectors expressing a V_(H)constant region, e.g., the human gamma 4 constant region, and the PCRamplified V_(L) domains can be cloned into vectors expressing a V₁constant region, e.g., human kappa or lamba constant regions.Preferably, the vectors for expressing the V_(H) or V_(L) domainscomprise an EF-1α promoter, a secretion signal, a cloning site for thevariable domain, constant domains, and a selection marker such asneomycin. The V_(H) and V_(L) domains may also be cloned into one vectorexpressing the necessary constant regions. The heavy chain conversionvectors and light chain conversion vectors are then co-transfected intocell lines to generate stable or transient cell lines that expressfull-length antibodies, e.g., IgG, using techniques known to those ofskill in the art.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison etal., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

The anti-FAP antibodies described herein specifically include chimericantibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while another portion of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).Chimeric antibodies of interest herein include “primatized” antibodiescomprising variable domain antigen-binding sequences derived from anonhuman primate (e.g., Old World Monkey, such as baboon, rhesus orcynomolgus monkey) and human constant region sequences (U.S. Pat. No.5,693,780).

A humanized antibody can be produced using a variety of techniques knownin the art, including but not limited to, CDR-grafting (see, e.g.,European Patent No. EP 239,400; International Publication No. WO91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, eachof which is incorporated herein in its entirety by reference), veneeringor resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnickaet al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al.,1994, PNAS, 91:969-973, each of which is incorporated herein by itsentirety by reference), chain shuffling (see, e.g., U.S. Pat. No.5,565,332, which is incorporated herein in its entirety by reference),and techniques disclosed in, e.g., published U.S. patent applicationUS2005/0042664, published U.S. patent application US2005/0048617, U.S.Pat. Nos. 6,407,213, 5,766,886, International Publication No. WO9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al.,Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79(2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska etal., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55(23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22(1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J.Mol. Biol., 235(3):959-73 (1994), each of which is incorporated hereinin its entirety by reference. Often, framework residues in the frameworkregions will be substituted with the corresponding residue from the CDRdonor antibody to alter, preferably improve, antigen binding. Theseframework substitutions are identified by methods well known in the art,e.g., by modeling of the interactions of the CDR and framework residuesto identify framework residues important for antigen binding andsequence comparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; andRiechmann et al., 1988, Nature, 332:323, which are incorporated hereinby reference in their entireties.)

A humanized anti-FAP antibody has one or more amino acid residuesintroduced into it from a source which is nonhuman. These nonhuman aminoacid residues are often referred to as “import” residues, which aretypically taken from an “import” variable domain. Thus, humanizedantibodies comprise one or more CDRs from nonhuman immunoglobulinmolecules and framework regions from human. Humanization of antibodiesis well known in the art and can essentially be performed following themethod of Winter and co-workers (Jones et al., Nature, 321:522-525(1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al.,Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody, i.e.,CDR-grafting, (EP 239,400; PCT Publication No. WO 91/09967; and U.S.Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089;6,548,640, the contents of which are incorporated herein by referenceherein in their entirety). In such humanized chimeric antibodies,substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a nonhuman species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies. Humanization ofanti-FAP antibodies can also be achieved by veneering or resurfacing (EP592,106; EP 519,596; Padlan, 1991, Molecular Immunology,28(4/5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814(1994); and Roguska et al., PNAS, 91:969-973 (1994)) or chain shuffling(U.S. Pat. No. 5,565,332), the contents of which are incorporated hereinby reference herein in their entirety. Anti-FAP antibodies can behumanized with retention of high affinity for FAP and other favorablebiological properties. A “humanized” antibody retains a similarantigenic specificity as the original antibody, i.e., in the presentinvention, the ability to bind human FAP antigen. However, using certainmethods of humanization, the affinity and/or specificity of binding ofthe antibody for human FAP antigen may be increased using methods of“directed evolution”, as described by Wu et al., J. Mol. Biol., 294:151(1999), the contents of which are incorporated herein by referenceherein in their entirety.

For in vivo use of antibodies in humans, it may be preferable to usehuman antibodies. Completely human antibodies are particularly desirablefor therapeutic treatment of human subjects. Human antibodies can bemade by a variety of methods known in the art including phage displaymethods described above using antibody libraries derived from humanimmunoglobulin sequences, including improvements to these techniques.See also U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO96/33735, and WO 91/10741; each of which is incorporated herein byreference in its entirety. A human antibody can also be an antibodywherein the heavy and light chains are encoded by a nucleotide sequencederived from one or more sources of human DNA.

Human anti-FAP antibodies can also be produced using transgenic micewhich are incapable of expressing functional endogenous immunoglobulins,but which can express human immunoglobulin genes. Human antibodies canalso be derived from phage-display libraries (Hoogenboom et al., J. Mol.Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991);Vaughan et al., Nature Biotech., 14:309 (1996)). Phage displaytechnology (McCafferty et al., Nature, 348:552-553 (1990)) can be usedto produce human antibodies and antibody fragments in vitro, fromimmunoglobulin variable (V) domain gene repertoires from unimmunizeddonors. Human antibodies may also be generated by in vitro activated Bcells (see, U.S. Pat. Nos. 5,567,610 and 5,229,275, each of which isincorporated herein by reference in its entirety). Human antibodies mayalso be generated by in vitro using hybridoma techniques such as, butnot limited to, that described by Roder et al. (Methods Enzymol.,121:140-167 (1986)).

“Antibody fragments” comprise a portion of a full-length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Traditionally, these fragments were derived via proteolytic digestion ofintact antibodies (see, e.g., Morimoto et al., Journal of Biochemicaland Biophysical Methods, 24:107-117 (1992) and Brennan et al., Science,229:81 (1985)). However, these fragments can now be produced directly byrecombinant host cells. For example, the antibody fragments can beisolated from the antibody phage libraries discussed above.Alternatively, Fab′-SH fragments can be directly recovered from E. coliand chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology, 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See, forexample, WO 93/16185. In certain embodiments, the antibody is not a Fabfragment.

Therapeutic Anti-FAP Compositions

The anti-FAP antibody used in the compositions and methods of theinvention is preferably an antibody that mediates the specificelimination of FAP expressing cells. The antibody may be a polyclonalantibody, monoclonal antibody, synthetic antibody, humanized antibody,human antibody, or fragments thereof.

The anti-FAP antibodies used in the compositions and methods of theinvention can be naked antibodies, immunoconjugates, chimeric antigenreceptors, or fusion proteins. Preferably the anti-FAP antibodiesdescribed above for use in the compositions and methods of the inventionare able to reduce or deplete stromal cells in a human treatedtherewith. Such depletion may be achieved via various mechanisms such asantibody-dependent cell-mediated cytotoxicity (ADCC) and/or complementdependent cytotoxicity (CDC). By “depletion” of stromal cells it ismeant a reduction in stromal cells in particular tissue(s) by at leastabout 25%, 40%, 50%, 65%, 75%, 80%, 85%, 90%, 95% or more. In particularembodiments, virtually all detectable stromal cells are depleted fromthe particular tissue(s).

Covalent modifications of the anti-FAP antibody of the invention areincluded within the scope of this invention. They may be made bychemical synthesis or by enzymatic or chemical cleavage of the antibody,if applicable. Other types of covalent modifications of the anti-FAPantibody are introduced into the molecule by reacting targeted aminoacid residues of the antibody with an organic derivatizing agent that iscapable of reacting with selected side chains or the N- or C-terminalresidues.

An anti-FAP antibody composition may be formulated with apharmaceutically-acceptable carrier. The term “pharmaceuticallyacceptable” means one or more non-toxic materials that do not interferewith the effectiveness of the biological activity of the activeingredients. Such preparations may routinely contain salts, bufferingagents, preservatives, compatible carriers, and optionally othertherapeutic agents. Such pharmaceutically acceptable preparations mayalso routinely contain compatible solid or liquid fillers, diluents orencapsulating substances which are suitable for administration into ahuman. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically-acceptable salts thereof and are notexcluded from the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, boric, formic,malonic, succinic, and the like. Also, pharmaceutically-acceptable saltscan be prepared as alkaline metal or alkaline earth salts, such assodium, potassium or calcium salts. The term “carrier” denotes anorganic or inorganic ingredient, natural or synthetic, with which theactive ingredient is combined to facilitate the application. Thecomponents of the pharmaceutical compositions also are capable of beingco-mingled with the antibodies of the present invention, and with eachother, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

The anti-FAP antibody compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide an immunosuppressiveagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The formulations to be used for in vivo administration are typicallysterile. This is readily accomplished by filtration through sterilefiltration membranes.

Administration of the compositions of the invention to a human patientcan be by any route, including but not limited to intravenous,intradermal, transdermal, subcutaneous, intramuscular, inhalation (e.g.,via an aerosol), buccal (e.g., sub-lingual), topical (i.e., both skinand mucosal surfaces, including airway surfaces), intrathecal,intraarticular, intraplural, intracerebral, intra-arterial,intraperitoneal, oral, intralymphatic, intranasal, rectal or vaginaladministration, by perfusion through a regional catheter, or by directintralesional injection. In a preferred embodiment, the compositions ofthe invention are administered by intravenous push or intravenousinfusion given over defined period (e.g., 0.5 to 2 hours). Thecompositions of the invention can be delivered by peristaltic means orin the form of a depot, although the most suitable route in any givencase will depend, as is well known in the art, on such factors as thespecies, age, gender and overall condition of the subject, the natureand severity of the condition being treated and/or on the nature of theparticular composition (i.e., dosage, formulation) that is beingadministered. In particular embodiments, the route of administration isvia bolus or continuous infusion over a period of time, once or twice aweek. In other particular embodiments, the route of administration is bysubcutaneous injection given in one or more sites (e.g. thigh, waist,buttocks, arm), optionally once or twice weekly. In one embodiment, thecompositions, and/or methods of the invention are administered on anoutpatient basis.

In certain embodiments, the dose of a composition comprising anti-FAPantibody is measured in units of mg/kg of patient body weight. In otherembodiments, the dose of a composition comprising anti-FAP antibody ismeasured in units of mg/kg of patient lean body weight (i.e., bodyweight minus body fat content). In yet other embodiments, the dose of acomposition comprising anti-FAP antibody is measured in units of mg/m²of patient body surface area. In yet other embodiments, the dose of acomposition comprising anti-FAP antibody is measured in units of mg perdose administered to a patient. Any measurement of dose can be used inconjunction with the compositions and methods of the invention anddosage units can be converted by means standard in the art.

Those skilled in the art will appreciate that dosages can be selectedbased on a number of factors including the age, sex, species andcondition of the subject (e.g., activity of autoimmune disease ordisorder), the desired degree of cellular or autoimmune antibodydepletion, the disease to be treated and/or the particular antibody orantigen-binding fragment being used and can be determined by one ofskill in the art. For example, effective amounts of the compositions ofthe invention may be extrapolated from dose-response curves derived fromin vitro test systems or from animal model (e.g. the cotton rat ormonkey) test systems. Models and methods for evaluation of the effectsof antibodies are known in the art (Wooldridge et al., Blood, 89(8):2994-2998 (1997), incorporated by reference herein in its entirety). Incertain embodiments, for a particular disease or disorder, therapeuticregimens standard in the art for antibody therapy can be used with thecompositions and methods of the invention.

Examples of dosing regimens that can be used in the methods of theinvention include, but are not limited to, daily, three times weekly(intermittent), weekly, or every 14 days. In certain embodiments, dosingregimens include, but are not limited to, monthly dosing or dosing every6-8 weeks. Those skilled in the art will appreciate that dosages aregenerally higher and/or frequency of administration greater for initialtreatment as compared with maintenance regimens.

CARs

The present invention provides a chimeric antigen receptor (CAR)comprising an extracellular and intracellular domain. The extracellulardomain comprises a target-specific binding element otherwise referred toas an antigen binding domain. In some embodiments, the extracellulardomain also comprises a hinge domain. The intracellular domain orotherwise the cytoplasmic domain comprises, a costimulatory signalingregion and a zeta chain portion. The costimulatory signaling regionrefers to a portion of the CAR comprising the intracellular domain of acostimulatory molecule. Costimulatory molecules are cell surfacemolecules other than antigens receptors or their ligands that arerequired for an efficient response of lymphocytes to antigen.

Between the extracellular domain and the transmembrane domain of theCAR, or between the cytoplasmic domain and the transmembrane domain ofthe CAR, there may be incorporated a spacer domain. As used herein, theterm “spacer domain” generally means any oligo- or polypeptide thatfunctions to link the transmembrane domain to, either the extracellulardomain or, the cytoplasmic domain in the polypeptide chain. A spacerdomain may comprise up to 300 amino acids, preferably 10 to 100 aminoacids and most preferably 25 to 50 amino acids.

The present invention includes retroviral and lentiviral vectorconstructs expressing a CAR that can be directly transduced into a cell.The present invention also includes an RNA construct that can bedirectly transfected into a cell. A method for generating mRNA for usein transfection involves in vitro transcription (IVT) of a template withspecially designed primers, followed by polyA addition, to produce aconstruct containing 3′ and 5′ untranslated sequence (“UTR”), a 5′ capand/or Internal Ribosome Entry Site (IRES), the gene to be expressed,and a polyA tail, typically 50-2000 bases in length. RNA so produced canefficiently transfect different kinds of cells. In one embodiment, thetemplate includes sequences for the CAR.

Preferably, the CAR comprises an extracellular domain, a transmembranedomain and a cytoplasmic domain. The extracellular domain andtransmembrane domain can be derived from any desired source of suchdomains. In one embodiment, the CAR comprises a nucleic acid sequencecomprising SEQ ID NO: 1. In one embodiment, the CAR comprises an aminoacid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:1.

Antigen Binding Domain

The extracellular domain may be obtained from any of the wide variety ofextracellular domains or secreted proteins associated with ligandbinding and/or signal transduction. In one embodiment, the extracellulardomain may consist of an Ig heavy chain which may in turn be covalentlyassociated with Ig light chain by virtue of the presence of CH1 andhinge regions, or may become covalently associated with other Igheavy/light chain complexes by virtue of the presence of hinge, CH2 andCH3 domains. In the latter case, the heavy/light chain complex thatbecomes joined to the chimeric construct may constitute an antibody witha specificity distinct from the antibody specificity of the chimericconstruct. Depending on the function of the antibody, the desiredstructure and the signal transduction, the entire chain may be used or atruncated chain may be used, where all or a part of the CH1, CH2, or CH3domains may be removed or all or part of the hinge region may beremoved.

The present invention comprises an antigen binding domain that binds toa stromal cell antigen. As discussed elsewhere herein, the presentinvention provides that targeting of the stromal cells existing in thein the tumor microenvironment allows for the reduction and/orelimination of the tumor. In one embodiment, the antigen binding domaincomprises a domain directed to FAP. FAP is expressed on a vast majorityof stromal cells in many types of human carcinomas. In one embodiment,the CAR may be one for which a specific monoclonal antibody currentlyexists or can be generated in the future. The tumor may be of any type,wherein the tumor microenvironment includes stromal cells. In oneembodiment, the tumor is a carcinoma.

In one embodiment, the retroviral or lentiviral vector comprises a CARdesigned to be directed to FAP by way of engineering an anti-FAP domaininto the CAR. In another embodiment, the template for the RNA CAR isdesigned to be directed to FAP by way of engineering an anti-FAP domaininto the CAR. The CAR of the invention can be engineered to include anyanti-FAP moiety that is specific to FAP. The antigen binding domain canbe any domain that binds to the antigen including but not limited tomonoclonal antibodies, polyclonal antibodies, synthetic antibodies,scFvs, human antibodies, humanized antibodies, and fragments thereof. Inone embodiment, the antigen binding domain comprises a nucleic acidsequence comprising SEQ ID NO: 3. In one embodiment, the antigen bindingdomain comprises an amino acid sequence encoded by a nucleic acidsequence comprising SEQ ID NO: 3.

Transmembrane Domain

With respect to the transmembrane domain, the CAR can be designed tocomprise a transmembrane domain that is fused to the extracellulardomain of the CAR. In one embodiment, the transmembrane domain thatnaturally is associated with one of the domains in the CAR is used. Insome instances, the transmembrane domain can be selected or modified byamino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use in this invention may be derived from (i.e. compriseat least the transmembrane region(s) of) the alpha, beta or zeta chainof the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.Alternatively the transmembrane domain may be synthetic, in which caseit will comprise predominantly hydrophobic residues such as leucine andvaline. Preferably a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.Optionally, a short oligo- or polypeptide linker, preferably between 2and 10 amino acids in length may form the linkage between thetransmembrane domain and the cytoplasmic signaling domain of the CAR. Aglycine-serine doublet provides a particularly suitable linker. In oneembodiment, the transmembrane domain comprises a nucleic acid sequencecomprising SEQ ID NO: 5. In one embodiment, the transmembrane domaincomprises an amino acid sequence encoded by a nucleic acid sequencecomprising SEQ ID NO: 5.

Cytoplasmic Domain

The cytoplasmic domain or otherwise the intracellular signaling domainof the CAR of the invention is responsible for activation of at leastone of the normal effector functions of the immune cell in which the CARhas been placed in. The term “effector function” refers to a specializedfunction of a cell. Effector function of a T cell, for example, may becytolytic activity or helper activity including the secretion ofcytokines. Thus the term “intracellular signaling domain” refers to theportion of a protein which transduces the effector function signal anddirects the cell to perform a specialized function. While usually theentire intracellular signaling domain can be employed, in many cases itis not necessary to use the entire chain. To the extent that a truncatedportion of the intracellular signaling domain is used, such truncatedportion may be used in place of the intact chain as long as ittransduces the effector function signal. The term intracellularsignaling domain is thus meant to include any truncated portion of theintracellular signaling domain sufficient to transduce the effectorfunction signal.

Preferred examples of intracellular signaling domains for use in the CARof the invention include the cytoplasmic sequences of the T cellreceptor (TCR) and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivative or variant of these sequences and any synthetic sequence thathas the same functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of cytoplasmic signalingsequence: those that initiate antigen-dependent primary activationthrough the TCR (primary cytoplasmic signaling sequences) and those thatact in an antigen-independent manner to provide a secondary orco-stimulatory signal (secondary cytoplasmic signaling sequences).

Primary cytoplasmic signaling sequences regulate primary activation ofthe TCR complex either in a stimulatory way, or in an inhibitory way.Primary cytoplasmic signaling sequences that act in a stimulatory mannermay contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary cytoplasmic signaling sequences thatare of particular use in the invention include those derived from TCRzeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22,CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmicsignaling molecule in the CAR of the invention comprises a cytoplasmicsignaling sequence derived from CD3 zeta.

In a preferred embodiment, the cytoplasmic domain of the CAR can bedesigned to comprise the CD3-zeta signaling domain by itself or combinedwith any other desired cytoplasmic domain(s) useful in the context ofthe CAR of the invention. For example, the cytoplasmic domain of the CARcan comprise a CD3 zeta chain portion and a costimulatory signalingregion. The costimulatory signaling region refers to a portion of theCAR comprising the intracellular domain of a costimulatory molecule. Acostimulatory molecule is a cell surface molecule other than an antigenreceptor or their ligands that is required for an efficient response oflymphocytes to an antigen. Examples of such molecules include CD27,CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and a ligand that specifically binds with CD83, and the like. Thus,while the invention in exemplified primarily with 4-1BB as theco-stimulatory signaling element, other costimulatory elements arewithin the scope of the invention.

The cytoplasmic signaling sequences within the cytoplasmic signalingportion of the CAR of the invention may be linked to each other in arandom or specified order. Optionally, a short oligo- or polypeptidelinker, preferably between 2 and 10 amino acids in length may form thelinkage. A glycine-serine doublet provides a particularly suitablelinker.

In one embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28. In yetanother embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28 and 4-1BB.

In one embodiment, the cytoplasmic domain comprises a nucleic acidsequence comprising SEQ ID NO: 6. In one embodiment, the cytoplasmicdomain comprises a nucleic acid sequence comprising SEQ ID NO: 7. In oneembodiment, the cytoplasmic domain comprises a nucleic acid sequencecomprising SEQ ID NO: 6 and SEQ ID NO: 7

In one embodiment, the cytoplasmic domain comprises an amino acidsequence encoded by a nucleic acid sequence comprising SEQ ID NO: 6. Inone embodiment, the cytoplasmic domain comprises an amino acid sequenceencoded by a nucleic acid sequence comprising SEQ ID NO: 7. In oneembodiment, the cytoplasmic domain comprises an amino acid sequenceencoded by a nucleic acid sequence comprising SEQ ID NO: 6 and SEQ IDNO: 7.

Vectors

The present invention encompasses a DNA construct comprising thesequence of a CAR, wherein the sequence comprises the nucleic acidsequence of an antigen binding domain operably linked to the nucleicacid sequence of an intracellular domain. An exemplary intracellulardomain that can be used in the CAR of the invention includes but is notlimited to the intracellular domain of CD3-zeta, CD28, 4-1BB, and thelike. In some instances, the CAR can comprise any combination ofCD3-zeta, CD28, 4-1BB, and the like.

In one embodiment, the CAR of the invention comprises anti-FAP, humanCD8 hinge and transmembrane domain, and 4-1BB and CD3zeta signalingdomains. In one embodiment, the sequence of the DNA construct comprisesSEQ ID NO: 1.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

The present invention also provides vectors in which a DNA of thepresent invention is inserted. Vectors derived from retroviruses such asthe lentivirus are suitable tools to achieve long-term gene transfersince they allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CARs is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors canbe suitable for replication and integration eukaryotes. Typical cloningvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence.

The expression constructs of the present invention may also be used fornucleic acid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. In another embodiment, theinvention provides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

RNA Transfection

In one embodiment, the genetically modified T cells of the invention aremodified through the introduction of RNA. In one embodiment, an in vitrotranscribed RNA CAR can be introduced to a cell as a form of transienttransfection. The RNA is produced by in vitro transcription using apolymerase chain reaction (PCR)-generated template. DNA of interest fromany source can be directly converted by PCR into a template for in vitromRNA synthesis using appropriate primers and RNA polymerase. The sourceof the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA,cDNA, synthetic DNA sequence or any other appropriate source of DNA. Thedesired template for in vitro transcription is the CAR of the presentinvention. For example, the template for the RNA CAR comprises anextracellular domain comprising a FAP binding domain; a transmembranedomain comprising the hinge and transmembrane domain of CD8α; and acytoplasmic domain comprises the signaling domain of CD3-zeta and thesignaling domain of 4-1BB.

In one embodiment, the DNA to be used for PCR contains an open readingframe. The DNA can be from a naturally occurring DNA sequence from thegenome of an organism. In one embodiment, the DNA is a full length geneof interest of a portion of a gene. The gene can include some or all ofthe 5′ and/or 3′ untranslated regions (UTRs). The gene can include exonsand introns. In one embodiment, the DNA to be used for PCR is a humangene. In another embodiment, the DNA to be used for PCR is a human geneincluding the 5′ and 3′ UTRs. The DNA can alternatively be an artificialDNA sequence that is not normally expressed in a naturally occurringorganism. An exemplary artificial DNA sequence is one that containsportions of genes that are ligated together to form an open readingframe that encodes a fusion protein. The portions of DNA that areligated together can be from a single organism or from more than oneorganism.

Genes that can be used as sources of DNA for PCR include genes thatencode polypeptides that provide a therapeutic or prophylactic effect toan organism or that can be used to diagnose a disease or disorder in anorganism. Preferred genes are genes which are useful for a short termtreatment, or where there are safety concerns regarding dosage or theexpressed gene. For example, for treatment of cancer, autoimmunedisorders, parasitic, viral, bacterial, fungal or other infections, thetransgene(s) to be expressed may encode a polypeptide that functions asa ligand or receptor for cells of the immune system, or can function tostimulate or inhibit the immune system of an organism. In someembodiments, t is not desirable to have prolonged ongoing stimulation ofthe immune system, nor necessary to produce changes which last aftersuccessful treatment, since this may then elicit a new problem. Fortreatment of an autoimmune disorder, it may be desirable to inhibit orsuppress the immune system during a flare-up, but not long term, whichcould result in the patient becoming overly sensitive to an infection.

PCR is used to generate a template for in vitro transcription of mRNAwhich is used for transfection. Methods for performing PCR are wellknown in the art. Primers for use in PCR are designed to have regionsthat are substantially complementary to regions of the DNA to be used asa template for the PCR. “Substantially complementary”, as used herein,refers to sequences of nucleotides where a majority or all of the basesin the primer sequence are complementary, or one or more bases arenon-complementary, or mismatched. Substantially complementary sequencesare able to anneal or hybridize with the intended DNA target underannealing conditions used for PCR. The primers can be designed to besubstantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a genethat is normally transcribed in cells (the open reading frame),including 5′ and 3′ UTRs. The primers can also be designed to amplify aportion of a gene that encodes a particular domain of interest. In oneembodiment, the primers are designed to amplify the coding region of ahuman cDNA, including all or portions of the 5′ and 3′ UTRs. Primersuseful for PCR are generated by synthetic methods that are well known inthe art. “Forward primers” are primers that contain a region ofnucleotides that are substantially complementary to nucleotides on theDNA template that are upstream of the DNA sequence that is to beamplified. “Upstream” is used herein to refer to a location 5, to theDNA sequence to be amplified relative to the coding strand. “Reverseprimers” are primers that contain a region of nucleotides that aresubstantially complementary to a double-stranded DNA template that aredownstream of the DNA sequence that is to be amplified. “Downstream” isused herein to refer to a location 3′ to the DNA sequence to beamplified relative to the coding strand.

Any DNA polymerase useful for PCR can be used in the methods disclosedherein. The reagents and polymerase are commercially available from anumber of sources.

Chemical structures with the ability to promote stability and/ortranslation efficiency may also be used. The RNA preferably has 5′ and3′ UTRs. In one embodiment, the 5′ UTR is between zero and 3000nucleotides in length. The length of 5′ and 3′ UTR sequences to be addedto the coding region can be altered by different methods, including, butnot limited to, designing primers for PCR that anneal to differentregions of the UTRs. Using this approach, one of ordinary skill in theart can modify the 5′ and 3′ UTR lengths required to achieve optimaltranslation efficiency following transfection of the transcribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the gene of interest. Alternatively, UTR sequences that are notendogenous to the gene of interest can be added by incorporating the UTRsequences into the forward and reverse primers or by any othermodifications of the template. The use of UTR sequences that are notendogenous to the gene of interest can be useful for modifying thestability and/or translation efficiency of the RNA. For example, it isknown that AU-rich elements in 3′ UTR sequences can decrease thestability of mRNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous gene. Alternatively, when a 5′ UTR that is not endogenous tothe gene of interest is being added by PCR as described above, aconsensus Kozak sequence can be redesigned by adding the 5′ UTRsequence. Kozak sequences can increase the efficiency of translation ofsome RNA transcripts, but does not appear to be required for all RNAs toenable efficient translation. The requirement for Kozak sequences formany mRNAs is known in the art. In other embodiments the 5′ UTR can bederived from an RNA virus whose RNA genome is stable in cells. In otherembodiments various nucleotide analogues can be used in the 3′ or 5′ UTRto impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one preferred embodiment, the promoter isa T7 polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In a preferred embodiment, the mRNA has both a cap on the 5′ end and a3′ poly(A) tail which determine ribosome binding, initiation oftranslation and stability mRNA in the cell. On a circular DNA template,for instance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNAtemplate is molecular cloning. However polyA/T sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with polyA/T 3′stretch without cloning highly desirable.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (size can be 50-5000 T), or after PCR by any other method,including, but not limited to, DNA ligation or in vitro recombination.Poly(A) tails also provide stability to RNAs and reduce theirdegradation. Generally, the length of a poly(A) tail positivelycorrelates with the stability of the transcribed RNA. In one embodiment,the poly(A) tail is between 100 and 5000 adenosines.

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotidesresults in about a two-fold increase in the translation efficiency ofthe RNA. Additionally, the attachment of different chemical groups tothe 3′ end can increase mRNA stability. Such attachment can containmodified/artificial nucleotides, aptamers and other compounds. Forexample, ATP analogs can be incorporated into the poly(A) tail usingpoly(A) polymerase. ATP analogs can further increase the stability ofthe RNA.

5′ caps on also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded.

RNA can be introduced into target cells using any of a number ofdifferent methods, for instance, commercially available methods whichinclude, but are not limited to, electroporation (Amaxa Nucleofector-II(Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (HarvardInstruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver,Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposomemediated transfection using lipofection, polymer encapsulation, peptidemediated transfection, or biolistic particle delivery systems such as“gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.,12(8):861-70 (2001).

Genetically Modified T Cells

In some embodiments, the CAR sequences are delivered into cells using aretroviral or lentiviral vector. CAR-expressing retroviral andlentiviral vectors can be delivered into different types of eukaryoticcells as well as into tissues and whole organisms using transduced cellsas carriers or cell-free local or systemic delivery of encapsulated,bound or naked vectors. The method used can be for any purpose wherestable expression is required or sufficient.

In other embodiments, the CAR sequences are delivered into cells usingin vitro transcribed mRNA. In vitro transcribed mRNA CAR can bedelivered into different types of eukaryotic cells as well as intotissues and whole organisms using transfected cells as carriers orcell-free local or systemic delivery of encapsulated, bound or nakedmRNA. The method used can be for any purpose where transient expressionis required or sufficient.

The disclosed methods can be applied to the modulation of T cellactivity in basic research and therapy, in the fields of cancer, stemcells, acute and chronic infections, and autoimmune diseases, includingthe assessment of the ability of the genetically modified T cell to killa target cancer cell.

The methods also provide the ability to control the level of expressionover a wide range by changing, for example, the promoter or the amountof input RNA, making it possible to individually regulate the expressionlevel. Furthermore, the PCR-based technique of mRNA production greatlyfacilitates the design of the chimeric receptor mRNAs with differentstructures and combination of their domains. For example, varying ofdifferent intracellular effector/costimulator domains on multiplechimeric receptors in the same cell allows determination of thestructure of the receptor combinations which assess the highest level ofcytotoxicity against multi-antigenic targets, and at the same timelowest cytotoxicity toward normal cells.

One advantage of RNA transfection methods of the invention is that RNAtransfection is essentially transient and a vector-free: An RNAtransgene can be delivered to a lymphocyte and expressed thereinfollowing a brief in vitro cell activation, as a minimal expressingcassette without the need for any additional viral sequences. Underthese conditions, integration of the transgene into the host cell genomeis unlikely. Cloning of cells is not necessary because of the efficiencyof transfection of the RNA and its ability to uniformly modify theentire lymphocyte population.

Genetic modification of T cells with in vitro-transcribed RNA (IVT-RNA)makes use of two different strategies both of which have beensuccessively tested in various animal models. Cells are transfected within vitro-transcribed RNA by means of lipofection or electroporation.Preferably, it is desirable to stabilize IVT-RNA using variousmodifications in order to achieve prolonged expression of transferredIVT-RNA.

Some IVT vectors are known in the literature which are utilized in astandardized manner as template for in vitro transcription and whichhave been genetically modified in such a way that stabilized RNAtranscripts are produced. Currently protocols used in the art are basedon a plasmid vector with the following structure: a 5′ RNA polymerasepromoter enabling RNA transcription, followed by a gene of interestwhich is flanked either 3′ and/or 5′ by untranslated regions (UTR), anda 3′ polyadenyl cassette containing 50-70 A nucleotides. Prior to invitro transcription, the circular plasmid is linearized downstream ofthe polyadenyl cassette by type II restriction enzymes (recognitionsequence corresponds to cleavage site). The polyadenyl cassette thuscorresponds to the later poly(A) sequence in the transcript. As a resultof this procedure, some nucleotides remain as part of the enzymecleavage site after linearization and extend or mask the poly(A)sequence at the 3′ end. It is not clear, whether this nonphysiologicaloverhang affects the amount of protein produced intracellularly fromsuch a construct.

RNA has several advantages over more traditional plasmid or viralapproaches. Gene expression from an RNA source does not requiretranscription and the protein product is produced rapidly after thetransfection. Further, since the RNA has to only gain access to thecytoplasm, rather than the nucleus, and therefore typical transfectionmethods result in an extremely high rate of transfection. In addition,plasmid based approaches require that the promoter driving theexpression of the gene of interest be active in the cells under study.

In another aspect, the RNA construct can be delivered into the cells byelectroporation. See, e.g., the formulations and methodology ofelectroporation of nucleic acid constructs into mammalian cells astaught in US 2004/0014645, US 2005/0052630A1, US 2005/0070841 A1, US2004/0059285A1, US 2004/0092907A1. The various parameters includingelectric field strength required for electroporation of any known celltype are generally known in the relevant research literature as well asnumerous patents and applications in the field. See e.g., U.S. Pat. Nos.6,678,556, 7,171,264, and U.S. Pat. No. 7,173,116. Apparatus fortherapeutic application of electroporation are available commercially,e.g., the MedPulser™ DNA Electroporation Therapy System(Inovio/Genetronics, San Diego, Calif.), and are described in patentssuch as U.S. Pat. Nos. 6,567,694; 6,516,223, 5,993,434, 6,181,964,6,241,701, and 6,233,482; electroporation may also be used fortransfection of cells in vitro as described e.g. in US20070128708A1.Electroporation may also be utilized to deliver nucleic acids into cellsin vitro. Accordingly, electroporation-mediated administration intocells of nucleic acids including expression constructs utilizing any ofthe many available devices and electroporation systems known to those ofskill in the art presents an exciting new means for delivering an RNA ofinterest to a target cell.

Sources of T Cells

Prior to expansion and genetic modification of the T cells of theinvention, a source of T cells is obtained from a subject. T cells canbe obtained from a number of sources, including peripheral bloodmononuclear cells, bone marrow, lymph node tissue, cord blood, thymustissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, and tumors. In certain embodiments of the presentinvention, any number of T cell lines available in the art, may be used.In certain embodiments of the present invention, T cells can be obtainedfrom a unit of blood collected from a subject using any number oftechniques known to the skilled artisan, such as Ficoll™ separation. Inone preferred embodiment, cells from the circulating blood of anindividual are obtained by apheresis. The apheresis product typicallycontains lymphocytes, including T cells, monocytes, granulocytes, Bcells, other nucleated white blood cells, red blood cells, andplatelets. In one embodiment, the cells collected by apheresis may bewashed to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. In oneembodiment of the invention, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations. Again, surprisingly, initial activation steps in theabsence of calcium lead to magnified activation. As those of ordinaryskill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5)according to the manufacturer's instructions. After washing, the cellsmay be resuspended in a variety of biocompatible buffers, such as, forexample, Ca²⁻-free, Mg²⁺-free PBS, PlasmaLyte A, or other salinesolution with or without buffer. Alternatively, the undesirablecomponents of the apheresis sample may be removed and the cells directlyresuspended in culture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient or bycounterflow centrifugal elutriation. A specific subpopulation of Tcells, such as CD3⁺, CD28⁻, CD4⁺, CD8⁻, CD45RA⁺, and CD45RO⁻ T cells,can be further isolated by positive or negative selection techniques.For example, in one embodiment, T cells are isolated by incubation withanti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS®M-450 CD3/CD28 T, for a time period sufficient for positive selection ofthe desired T cells. In one embodiment, the time period is about 30minutes. In a further embodiment, the time period ranges from 30 minutesto 36 hours or longer and all integer values there between. In a furtherembodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. Inyet another preferred embodiment, the time period is 10 to 24 hours. Inone preferred embodiment, the incubation time period is 24 hours. Forisolation of T cells from patients with leukemia, use of longerincubation times, such as 24 hours, can increase cell yield. Longerincubation times may be used to isolate T cells in any situation wherethere are few T cells as compared to other cell types, such in isolatingtumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmune-compromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainembodiments, it may be desirable to perform the selection procedure anduse the “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4⁻ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8. In certain embodiments, it may be desirable to enrichfor or positively select for regulatory T cells which typically expressCD4⁺, CD25⁺, CD62L^(hi), GITR⁺, and FoxP3⁺. Alternatively, in certainembodiments, T regulatory cells are depleted by anti-C25 conjugatedbeads or other similar method of selection.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (i.e., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8⁺ T cells that normally haveweaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4⁺ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8⁺ T cells in dilute concentrations. In one embodiment, theconcentration of cells used is 5×10⁶/ml. In other embodiments, theconcentration used can be from about 1×10⁵/ml to 1×10⁶/ml, and anyinteger value in between.

In other embodiments, the cells may be incubated on a rotator forvarying lengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

In certain embodiments, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in T cell therapy for any number of diseases or conditionsthat would benefit from T cell therapy, such as those described herein.In one embodiment a blood sample or an apheresis is taken from agenerally healthy subject. In certain embodiments, a blood sample or anapheresis is taken from a generally healthy subject who is at risk ofdeveloping a disease, but who has not yet developed a disease, and thecells of interest are isolated and frozen for later use. In certainembodiments, the T cells may be expanded, frozen, and used at a latertime. In certain embodiments, samples are collected from a patientshortly after diagnosis of a particular disease as described herein butprior to any treatments. In a further embodiment, the cells are isolatedfrom a blood sample or an apheresis from a subject prior to any numberof relevant treatment modalities, including but not limited to treatmentwith agents such as natalizumab, efalizumab, antiviral agents,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies,cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, FR901228, and irradiation. These drugs inhibit either thecalcium dependent phosphatase calcineurin (cyclosporine and FK506) orinhibit the p70S6 kinase that is important for growth factor inducedsignaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson etal., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun.5:763-773, 1993). In a further embodiment, the cells are isolated for apatient and frozen for later use in conjunction with (e.g., before,simultaneously or following) bone marrow or stem cell transplantation, Tcell ablative therapy using either chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, orantibodies such as OKT3 or CAMPATH. In another embodiment, the cells areisolated prior to and can be frozen for later use for treatmentfollowing B-cell ablative therapy such as agents that react with CD20,e.g., Rituxan.

In a further embodiment of the present invention, T cells are obtainedfrom a patient directly following treatment. In this regard, it has beenobserved that following certain cancer treatments, in particulartreatments with drugs that damage the immune system, shortly aftertreatment during the period when patients would normally be recoveringfrom the treatment, the quality of T cells obtained may be optimal orimproved for their ability to expand ex vivo. Likewise, following exvivo manipulation using the methods described herein, these cells may bein a preferred state for enhanced engraftment and in vivo expansion.Thus, it is contemplated within the context of the present invention tocollect blood cells, including T cells, dendritic cells, or other cellsof the hematopoietic lineage, during this recovery phase. Further, incertain embodiments, mobilization (for example, mobilization withGM-CSF) and conditioning regimens can be used to create a condition in asubject wherein repopulation, recirculation, regeneration, and/orexpansion of particular cell types is favored, especially during adefined window of time following therapy. Illustrative cell typesinclude T cells, B cells, dendritic cells, and other cells of the immunesystem.

Activation and Expansion of T Cells

Whether prior to or after genetic modification of the T cells to expressa desirable CAR, the T cells can be activated and expanded generallyusing methods as described, for example, in U.S. Pat. Nos. 6,352,694;6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681;7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223;6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application PublicationNo. 20060121005.

Generally, the T cells of the invention are expanded by contact with asurface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a co-stimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4⁺ T cells or CD8⁺ T cells, an anti-CD3 antibody and ananti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besançon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In certain embodiments, the primary stimulatory signal and theco-stimulatory signal for the T cell may be provided by differentprotocols. For example, the agents providing each signal may be insolution or coupled to a surface. When coupled to a surface, the agentsmay be coupled to the same surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentmay be coupled to a surface and the other agent in solution. In oneembodiment, the agent providing the co-stimulatory signal is bound to acell surface and the agent providing the primary activation signal is insolution or coupled to a surface. In certain embodiments, both agentscan be in solution. In another embodiment, the agents may be in solubleform, and then cross-linked to a surface, such as a cell expressing Fcreceptors or an antibody or other binding agent which will bind to theagents. In this regard, see for example, U.S. Patent ApplicationPublication Nos. 20040101519 and 20060034810 for artificial antigenpresenting cells (aAPCs) that are contemplated for use in activating andexpanding T cells in the present invention.

In one embodiment, the two agents are immobilized on beads, either onthe same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By wayof example, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the co-stimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one embodiment, a 1:1ratio of each antibody bound to the beads for CD4⁻ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular embodiment an increaseof from about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain embodiments ofthe invention, the ratio of anti CD28 antibody to anti CD3 antibodybound to the beads is greater than 2:1. In one particular embodiment, a1:100 CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. Ina further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beadsis used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody boundto beads is used. In one preferred embodiment, a 1:10 CD3:CD28 ratio ofantibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28ratio of antibody bound to the beads is used. In yet another embodiment,a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain embodiments the ratio of cellsto particles ranges from 1:100 to 100:1 and any integer valuesin-between and in further embodiments the ratio comprises 1:9 to 9:1 andany integer values in between, can also be used to stimulate T cells.The ratio of anti-CD3- and anti-CD28-coupled particles to T cells thatresult in T cell stimulation can vary as noted above, however certainpreferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, and 15:1 with one preferred ratio being at least 1:1particles per T cell. In one embodiment, a ratio of particles to cellsof 1:1 or less is used. In one particular embodiment, a preferredparticle: cell ratio is 1:5. In further embodiments, the ratio ofparticles to cells can be varied depending on the day of stimulation.For example, in one embodiment, the ratio of particles to cells is from1:1 to 10:1 on the first day and additional particles are added to thecells every day or every other day thereafter for up to 10 days, atfinal ratios of from 1:1 to 1:10 (based on cell counts on the day ofaddition). In one particular embodiment, the ratio of particles to cellsis 1:1 on the first day of stimulation and adjusted to 1:5 on the thirdand fifth days of stimulation. In another embodiment, particles areadded on a daily or every other day basis to a final ratio of 1:1 on thefirst day, and 1:5 on the third and fifth days of stimulation. Inanother embodiment, the ratio of particles to cells is 2:1 on the firstday of stimulation and adjusted to 1:10 on the third and fifth days ofstimulation. In another embodiment, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type.

In further embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application ofa force, such as a magnetic force, resulting in increased ligation ofcell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one embodiment the cells (for example,10⁴ to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer,preferably PBS (without divalent cations such as, calcium andmagnesium). Again, those of ordinary skill in the art can readilyappreciate any cell concentration may be used. For example, the targetcell may be very rare in the sample and comprise only 0.01% of thesample or the entire sample (i.e., 100%) may comprise the target cell ofinterest. Accordingly, any cell number is within the context of thepresent invention. In certain embodiments, it may be desirable tosignificantly decrease the volume in which particles and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and particles. For example, in one embodiment, aconcentration of about 2 billion cells/ml is used. In anotherembodiment, greater than 100 million cells/ml is used. In a furtherembodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,or 50 million cells/ml is used. In yet another embodiment, aconcentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mlis used. In further embodiments, concentrations of 125 or 150 millioncells/ml can be used. Using high concentrations can result in increasedcell yield, cell activation, and cell expansion. Further, use of highcell concentrations allows more efficient capture of cells that mayweakly express target antigens of interest, such as CD28-negative Tcells. Such populations of cells may have therapeutic value and would bedesirable to obtain in certain embodiments. For example, using highconcentration of cells allows more efficient selection of CD8+ T cellsthat normally have weaker CD28 expression.

In one embodiment of the present invention, the mixture may be culturedfor several hours (about 3 hours) to about 14 days or any hourly integervalue in between. In another embodiment, the mixture may be cultured for21 days. In one embodiment of the invention the beads and the T cellsare cultured together for about eight days. In another embodiment, thebeads and T cells are cultured together for 2-3 days. Several cycles ofstimulation may also be desired such that culture time of T cells can be60 days or more. Conditions appropriate for T cell culture include anappropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or,X-vivo 15, (Lonza)) that may contain factors necessary for proliferationand viability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, and TNF-α or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (T_(H), CD4⁺) that is greater than the cytotoxic orsuppressor T cell population (T_(C), CD8⁺). Ex vivo expansion of T cellsby stimulating CD3 and CD28 receptors produces a population of T cellsthat prior to about days 8-9 consists predominately of T_(H) cells,while after about days 8-9, the population of T cells comprises anincreasingly greater population of T_(C) cells. Accordingly, dependingon the purpose of treatment, infusing a subject with a T cell populationcomprising predominately of T_(H) cells may be advantageous. Similarly,if an antigen-specific subset of T_(C) cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Therapeutic Application

The present invention encompasses a cell (e.g., T cell) modified toexpress a CAR that combines an FAP binding domain with an intracellulardomain of CD3-zeta, CD28, 4-1BB, or any combinations thereof. Therefore,in some instances, the modified T cell can elicit a CAR-mediated T-cellresponse.

The invention provides the use of a CAR to redirect the specificity of aprimary T cell to a stromal cell antigen. Thus, the present inventionalso provides a method for stimulating a T cell-mediated immune responseto a population of stromal cells within a tumor microenvironment in amammal comprising the step of administering to the mammal a T cell thatexpresses a CAR, wherein the CAR comprises a binding moiety thatspecifically interacts with a predetermined stromal cell antigen, a zetachain portion comprising for example the intracellular domain of humanCD3zeta, and a costimulatory signaling region.

In one embodiment, the present invention includes a type of cellulartherapy where T cells are genetically modified to express a CAR and theCAR T cell is infused to a recipient in need thereof. The infused cellis able to reduce tumor burden in the recipient. Unlike antibodytherapies, CAR T cells are able to replicate in vivo resulting inlong-term persistence that can lead to sustained tumor control.

In one embodiment, the CAR T cells of the invention can undergo robustin vivo T cell expansion and can persist for an extended amount of time.In another embodiment, the CAR T cells of the invention evolve intospecific memory T cells that can be reactivated to inhibit anyadditional tumor formation or growth. For example, CAR T cells of theinvention can undergo robust in vivo T cell expansion and persist athigh levels for an extended amount of time in blood and bone marrow andform specific memory T cells. Without wishing to be bound by anyparticular theory, CAR T cells may differentiate in vivo into a centralmemory-like state upon encounter and subsequent elimination of targetcells expressing the surrogate antigen.

Without wishing to be bound by any particular theory, the responseelicited by the CAR-modified T cells may be an active or a passiveimmune response. In addition, the CAR mediated immune response may bepart of an adoptive immunotherapy approach in which CAR-modified T cellsinduce an immune response specific to the antigen binding domain in theCAR. For example, a FAP-CAR T cells elicits an immune response specificagainst cells expressing FAP.

While the data disclosed herein specifically disclose a CAR comprisingan anti-FAP binding domain, along with 4-1BB and CD3zeta signalingdomains, the invention should be construed to include any number ofvariations for each of the components of the construct as describedelsewhere herein. That is, the invention includes the use of any stromalcell antigen binding domain in the CAR to generate a CAR-mediated T-cellresponse specific to the antigen binding domain.

As described elsewhere herein, the present invention provides thetargeting of stromal cells which exist in the tumor microenvironment totreat cancers. As such, the present invention includes the treatment ofany cancer where stromal cells exist. Cancers that may be treatedinclude tumors that are not vascularized, or not yet substantiallyvascularized, as well as vascularized tumors. The cancers may comprisenon-solid tumors (such as hematological tumors, for example, leukemiasand lymphomas) or may comprise solid tumors. Types of cancers to betreated with the CARs of the invention include, but are not limited to,carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoidmalignancies, benign and malignant tumors, and malignancies e.g.,sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatrictumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

The CAR-modified T cells of the invention may also serve as a type ofvaccine for ex vivo immunization and/or in vivo therapy in a mammal.Preferably, the mammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAR tothe cells, and/or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (preferably ahuman) and genetically modified (i.e., transduced or transfected invitro) with a vector expressing a CAR disclosed herein. The CAR-modifiedcell can be administered to a mammalian recipient to provide atherapeutic benefit. The mammalian recipient may be a human and theCAR-modified cell can be autologous with respect to the recipient.Alternatively, the cells can be allogeneic, syngeneic or xenogeneic withrespect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAR-modified Tcells of the invention are used in the treatment of cancer. In certainembodiments, the cells of the invention are used in the treatment ofpatients at risk for developing cancer. Thus, the present inventionprovides methods for the treatment or prevention of cancer comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the CAR-modified T cells of the invention.

In one embodiment, the T cells modified to express a CAR directedagainst a stromal cell antigen (e.g. FAP) is administered asmonotherapy. However, in another embodiment, the T cells modified toexpress a CAR directed against a stromal cell antigen are administeredin a combination therapy. For example, in one embodiment, the stromalcell directed CAR T cells are administered to a mammal along with Tcells modified to express a tumor-directed CAR, wherein thetumor-directed CAR comprises an antigen binding domain that targets anytumor antigen.

In one embodiment, the T cells modified to express a CAR directedagainst a stromal cell antigen (e.g. FAP) is administered as combinationtherapy along with an antitumor vaccine as disclosed elsewhere herein.

The CAR-modified T cells of the present invention may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2 or other cytokines orcell populations. Briefly, pharmaceutical compositions of the presentinvention may comprise a target cell population as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. Such compositions maycomprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such as EDTA or glutathione;adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions ofthe present invention are preferably formulated for intravenousadministration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount,” “an anti-tumor effectiveamount,” “an tumor-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319:1676, 1988). The optimal dosage and treatment regime fora particular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it may be desired to administer activated Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom according to thepresent invention, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks. In certain embodiments, T cells can be activated from blooddraws of from 10 cc to 400 cc. In certain embodiments, T cells areactivated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc,80 cc, 90 cc, or 100 cc. Not to be bound by theory, using this multipleblood draw/multiple reinfusion protocol may serve to select out certainpopulations of T cells.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one embodiment, the T cell compositions of thepresent invention are administered to a patient by intradermal orsubcutaneous injection. In another embodiment, the T cell compositionsof the present invention are preferably administered by i.v. injection.The compositions of T cells may be injected directly into a tumor, lymphnode, or site of infection.

In certain embodiments of the present invention, cells activated andexpanded using the methods described herein, or other methods known inthe art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to treatment with agents such asantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further embodiments, the T cells of the invention may be used incombination with chemotherapy, radiation, immunosuppressive agents, suchas cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAM PATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun.73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). Ina further embodiment, the cell compositions of the present invention areadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cell compositions ofthe present invention are administered following B-cell ablative therapysuch as agents that react with CD20, e.g., Rituxan. For example, in oneembodiment, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain embodiments, following the transplant, subjects receive aninfusion of the expanded immune cells of the present invention. In anadditional embodiment, expanded cells are administered before orfollowing surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices.Strategies for CAR T cell dosing and scheduling have been discussed(Ertl et al, 2011, Cancer Res, 71:3175-81; Junghans, 2010, Journal ofTranslational Medicine, 8:55).

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1: Adoptive Transfer of Chimeric Antigen Receptor Expressing TCells to Diminish Fibroblast Activation Protein Expressing Tumor StromalCells

Human T Cells Targeting Mouse FAP have been Produced

A hybridoma producing an anti-mouse FAP antibody (clone 73.3) wasgenerated. The immunoglobulin heavy and light chains were sequenced andinserted into a set of plasmid cassettes that contained 2 tandemsignaling domains: 4-1BB and CD3-zeta.

A lentiviral vector with the murine (m) mFAP-CAR construct wasconstructed and transduced into human T cells that were then evaluatedfor the level of expression of the CAR and the ability to secretecytokines and specifically kill FAP expressing cells. FIG. 2 comparesthe response of untransduced T cells and T cells transduced with the FAPconstruct (mFAP-CAR T cells). The T cells were reacted for 4 hours at aneffector (T cell) to target (fibroblasts) ratio of 10:1 with either wildtype (WT) 3T3 cells (mouse fibroblasts that do not express FAP) or 3T3cells transduced to express murine FAP (3T3mFAP). The untransduced Tcells alone and mixed with WT 3T3 or 3T3mFAP cells and mFAP-CAR-T cellsalone or mixed with WT 3T3 all expressed undetectable or negligiblelevels of IFN-γ while mFAP-CAR-T cells interacted with 3T3mFAP cellsproduced high levels of IFN-γ (FIG. 2, upper panel). Similarly, specificcytoxicity by mFAP-CAR T cells of 3T3mFAP cells was induced (FIG. 2,lower panel). These data show that mFAP-CAR human T cells selectivelyand efficiently released IFN-γ and killed FAP-expressing cells but notFAP-negative 3T3 fibroblasts in vitro.

Mouse T Cells Targeting Mouse FAP have been Produced

As lentiviral transduction is not effective for mouse T cells, availableprotocols were adapted (Lee et al., 2009, Methods Mol Biol, 506: 83-96;Zhong et al., 2010, J Vis Exp, 44: 2307) to achieve high transductionrates using retroviral transduction. Accordingly, mouse T cells weretransduced with the mFAP-CAR-GFP retroviral vector. As shown in FIG. 3,transgene expression was observed in more than 70% of cells three daysafter transduction (with no selection) based on detection of GFP+ cellsby FACS of untransduced (UTD) vs. RV.CAR-GFP transduced cells (rightpanel). Similar results were seen when cells were stained with anantibody that recognizes the scFv portion of the CAR proving surfaceexpression. Thirty million CAR+ T cells are routinely generated perdonor spleen.

Anti-Tumor Activity of FAP-CAR T in Mouse Models:

A syngeneic transplant model of the mesothelioma AE17 (AE17) inimmunocompetent mice was used to test the anti-tumor activity ofmFAP-CAR T cells in vivo. An important feature of this model is that thetumors become infiltrated with FAP+ fibroblasts. FIG. 4 shows a mousetumor stained with anti-FAP antibody. This figure shows that hostfibroblasts infiltrate the tumors and that the FAP+ cells can be easilyidentified by immunostaining.

A study was then conducted to establish the safety and feasibility ofthe FAP-CAR approach. Mouse AE17 cells were injected into the flanks ofB6 mice and allowed to grow to 150 mm³ in size. The mice were injectedintravenously with saline (AE17), with 10⁷ mouse T cells transfectedwith a retrovirus encoding GFP (MigR1) or with 10⁷ mouse T cellstransfected with mFAP-CAR (H2L) (all were at 60% transfectionefficiency). FIG. 5 shows the tumor sizes 9 days after injection. Thissingle dose of mouse FAP-CAR T cells clearly inhibited tumor growth.Importantly, no toxicity in the mice was noted. These data demonstratethat mFAP-CAR T cells can inhibit tumor growth without obvious toxicity.

A study was conducted to show the safety and feasibility of the FAP CARapproach using human T cells. Human lung cancer cells (A549) wereinjected into the flanks of immunodeficient mice and allowed to grow to100 mm³ in size. The mice were injected intravenously with saline(untreated, or with 10⁷ human T cells transfected with a lentivirusexpressing mFAP-CAR (WT-H2L). FIG. 6 shows the tumor sizes before andafter injection (arrow). This single dose of human T cells expressingmouse FAP-CAR clearly inhibited tumor growth. Importantly, no toxicityin the mice was noted. These data demonstrate that mFAP-CAR in human Tcells can inhibit tumor growth without obvious toxicity.

Another study was performed to examine FAP-CAR T cell specificity. Tumorcells were injected into wild type mice or FAP-Knockout mice as aboveand treated with 10⁷ control T cells or with 10⁷ FAP-CAR T cells. FIG. 7shows the tumor sizes at the time of injection (Day 0) and at 4 and 8days after injection. While FAP-CAR T cells slowed tumor growth in wildtype mice (upper panel), all activity was lost in the FAP KO mice. Thesedata show that mFAP-CAR T cells can inhibit tumor growth without obvioustoxicity in mice and in a FAP-specific fashion (all activity was lost inFAP-KO mice).

As described herein, mFAP-CAR T cells have been developed that arespecifically activated by FAP expressing stromal cells in vitro andexhibit anti-tumor activity in vivo. It is demonstrated that mFAP-CAR Tcells are specifically activated by and kill FAP expressing stromalcells and have anti-tumor activity in a mouse model of cancer with noobvious toxicity.

Human T Cells Targeting Human-FAP have been Produced.

In a similar fashion as above, an anti-human FAP construct is optimized.The light and heavy chains of the well-characterized F19 anti-human FAPmonoclonal (the same antibody used to stain human lung cancer in FIG. 2)from hybridoma cells has been sequenced and inserted into theCD3-zeta:41BB “double activation domain” construct. Human T cells aretransduced with a humanFAP-CAR construct thereby allowing specifictargeting of human FAP.

The FAP-CAR approach presented herein is further evaluated by way ofadditional experiments. The specificity and anti-tumor activity of mouseand human FAP-CAR-T cells in human tumor xenografts in immunoincompetentmice is examined. Further animal and human studies are performed toexamine the ability of FAP-CAR T cells to inhibit metastasis. Additionalstudies are done to evaluate FAP-CAR T cells administered both as amonotherapy and also as a combined therapy along with tumor-directedCARs to observe any additive or synergistic effects. Studies presentedherein provide avenues to understand the mechanisms by which deletion ofFAP+ stromal cells inhibit primary tumor growth.

Example 2: Sequence of the mFAP-CAR

A vector map of the vector, pELNS-muFAP(H2toL)-CD8H-BBz, is shown inFIG. 8. The nucleic acid sequence of the mFAP-CAR (SEQ ID NO: 1) and ofindividual domains within the CAR are provided.

mFAP-CAR  (SEQ ID NO: 1)atggccctgcctgtgacagccctgctgctgcctctggctctgctgctgcatgccgctagacctggatcccaggtgcagctgaaagagtccggcggaggactggtgcagcctggcggatctctgaagctgagctgtgctgccagcggatcaccttcagcagctacggcatgagctgggtgcgacagaccgccgacaagagactggaactggtggctaccaccaacaacaacggcggcgtgacctactaccccgacagcgtgaagggcagattcaccatctccagagacaacgccaagaacaccctgtacctgcagatgagcagcctgcagagcgaggacaccgccatgtactactgcgccagatacggctactacgccatggattactggggccagggcatcagcgtgaccgtgtctagcggaggcggcggatctggcggagggggatctagtggcggaggctctgacgtgctgatgacccagacacctctgagcctgccagtgtccctgggcgaccaggccagcatcagctgtagaagcagccagagcatcgtgcacagcaacggcaacacctacctggaatggtatctgcagaagcccggccagagccccaagctgctgatctacaaggtgtccaacagattcagcggcgtgcccgacagattctccggcagcggctctggcaccgacttcaccgtgaagatctccagggtggaagccgaggacctgggcgtgtactactgttttcaaggcagccacgtgccctacaccttcggcggaggcaccaagctggaaatcaaggctagctccggaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc CD8 leader sequence  (SEQ ID NO: 2)atggccctgcctgtgacagccctgctgctgcctctggctctgctgctgca tgccgctagacctAnti-mFAP scFv  (SEQ ID NO: 3)caggtgcagctgaaagagtccggcggaggactggtgcagcctggcggatctctgaagctgagctgtgctgccagcggcttcaccttcagcagctacggcatgagctgggtgcgacagaccgccgacaagagactggaactggtggctaccaccaacaacaacggcggcgtgacctactaccccgacagcgtgaagggcagattcaccatctccagagacaacgccaagaacaccctgtacctgcagatgagcagcctgcagagcgaggacaccgccatgtactactgcgccagatacggctactacgccatggattactggggccagggcatcagcgtgaccgtgtctagcggaggcggcggatctggcggagggggatctagtggcggaggctctgacgtgctgatgacccagacacctctgagcctgccagtgtccctgggcgaccaggccagcatcagctgtagaagcagccagagcatcgtgcacagcaacggcaacacctacctggaatggtatctgcagaagcccggccagagccccaagctgctgatctacaaggtgtccaacagattcagcggcgtgcccgacagattctccggcagcggctctggcaccgacttcaccgtgaagatctccagggtggaagccgaggacctgggcgtgtactactgttttcaaggcagccacgtgccctacaccttcggcggaggcaccaagctggaaatcaag CD8a hinge  (SEQ ID NO: 4)accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat CD8a transmembrane domain (SEQ ID NO: 5) atctacatctgggcgccatggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc 4-1BB intracellular domain  (SEQ ID NO: 6)aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg CD3-zeta signaling domain  (SEQ ID NO: 7)Agagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc

Example 3: CAR T Cells Targeting FAP in the Tumor Stroma have AntitumorEfficacy and Augment Host Immunity Without Toxicity

As described elsewhere herein and in the present Example, a single chainFv specific for mouse FAP was expressed in a retroviral CAR constructcontaining a CD8α hinge and transmembrane regions and human CD3Z and4-1BB activation domains. Retrovirally-transduced muFAP-CAR mouse Tcells secreted IFNy and killed FAP-expressing 3T3 target cells, but didnot react with FAP-negative parental 3T3 cells. Adoptively transferredFAP-CAR mouse T cells reduced the number of FAP⁺ stromal cells andinhibited growth of multiple types of subcutaneously transplanted tumorsin wild-type, but not FAP-null immune-competent syngeneic mice. Theanti-tumor effects could be augmented by multiple injections of T cells,by using T cells with enhanced anti-tumor activity due to the loss ofdiacylglycerol kinase, or by combination with a vaccine. A majormechanism of action was augmentation of endogenous CD8+ T cellanti-tumor responses. Importantly, off-tumor toxicity was minimalfollowing muFAP-CAR T cell therapy. Therefore, inhibiting tumor growthby targeting tumor stroma with adoptively transferred CAR T cellsdirected to FAP has been discovered in the present invention to be afeasible, safe, and effective antitumor therapy.

The anti-FAP scFv used herein is different from that used in prior artstudies in that the antibody comprises both human and murine cytoplasmicdomains. The data presented herein establish clear anti-tumor efficacyin multiple tumor models, evidence of activation of endogenous immuneresponses, and success in combining the CAR T cells with a tumorvaccine. Importantly, using the CAR constructs of the present invention,minimal toxicity was apparent, with no accompanying anemia or weightloss.

The Materials and Methods used in the experiments described in thisExample are now described.

Cell Lines

Mouse AE17.ova mesothelioma cells (expressing chicken ovalbumin) wereused (Jackaman et al., 2003J Immunol. 171:5051-63). TC1 lung cancercells were derived from mouse lung epithelial cells immortalized withhuman papillomavirus HPV-16 E6 and E7 and transformed with the c-Ha-rasoncogene (Lin et al., 1996, Cancer Res 56:21-6). The mouse “LKR” cellline was derived from an explant of a pulmonary tumor from an activatedK-ras^(G12) ^(o) mutant mouse generated as described in Johnson et al.,2001, Nature 410:1111-6. Mouse 4T1 mammary carcinoma, CT26 colon cancercells and 3T3Balb/C cells, were purchased from the American Type CultureCollection. Mouse FAP expressing 3T3BALB/C (3T3.FAP) cells were createdby lentiviral transduction of the FAP-3T3 parental line with murine FAP(FIGS. 9A and 9B).

Antibodies

Specific antibodies used are described elsewhere herein.

Generation of 73.3 Hybridoma

FAP-null mice (Niedermeyer et al., 2000, Mol Cell Biol 2000;20:1089-94), were immunized and twice boosted with FAP+ 3T3 cellsintraperitoneally. 72 hours after the final boost, splenocytes wereharvested and fused to myeloma cells. Hybridoma supernatants werescreened for monoclonal antibodies that reacted specifically with3T3.FAP and activated primary wild-type fibroblasts but not 3T3 cells oractivated primary fibroblasts isolated from FAP-null mice. One mAb,73.3, with this reactivity profile was purified and furthercharacterized as specific for mouse FAP based on reactivity withpurified recombinant mouse FAP extracellular domain (by ELISA and byimmunoblotting), its reactivity with a single protein with an apparentmolecular weight of approximately 95 kD in protein extracts of FAP+ butnot FAP⁻ cells and tissues, and lack of reactivity with cells expressinghuman FAP (human FAP transduced 3T3 and human foreskin fibroblasts)(FIGS. 10A-10C).

Synthesis of Anti-muFAP CAR Construct.

Total RNA from 73.3 hybridoma cells was isolated and reverse transcribedto cDNA. Variable heavy (V_(H)) and light (V_(L)) chains of 73.3anti-muFAP antibody were PCR amplified and sequenced (FIGS. 11A and11B). The VH and VL sequences were fused with a CAR construct comprisingCD8a hinge, CD8a transmembrane domain, and two human intracellularsignaling domains derived from 4-1BB and CD3Q (Milone et al., 2009, MolTher. 17:1453-64). This CAR was then inserted into an IRES containingretroviral MigR1 vector (FIG. 11C) that also expresses green fluorescentprotein (GFP) for tracking purposes (Pear et al., 1998, Blood92(10):3780-92. A fully mouse construct of FAP-CAR, “FAP-CAR-m28Z”, wasalso synthesized by coupling the same 73.3 scFv with the murine CD3Zchain and murine CD28 intracellular signaling domain. This construct wasthen inserted into another retroviral vector MSGV (FIG. 11D) (Morgan etal., 2012, 23:1043-53). Infective particles were generated from thesupernatants of 293T cells transfected with retroviral vector plasmidand helper plasmids using Lipofectamine 2000 (Invitrogen), as described(Lee et al., m, 2009, Methods Mol Biol 506:83-96).

Isolation, Transduction and Expansion of Primary Mouse T lymphocytes

Primary murine splenic T cells were isolated and transduced as describedin Riese et al., 2013, Cancer Res 73:3566-77 and elsewhere herein.

Antigen or Antibody Coated Beads

Recombinant FAP-extracellular domain protein (FAP-ECD, bovine serumalbumin (Fisher Scientific) or anti-CD3e/anti-CD28 antibodies(eBioscience) were chemically crosslinked to tosylactivated 4.5 ^mDynabeads (Invitrogen, #140-13) using the manufacturers' instructions.

Immunoblotting

To assess the function of the FAP-CAR construct, FAP-CAR transduced Tcells were incubated either with BSA or FAP-ECD-coated beads (at 2:1bead to T cell ratio), or with anti-CD3e antibody for 10 min. Lysateswere then prepared and immunoblotted for phosphorylated ERK,phosphorylated AKT, phosphorylated IKKa/p, or P-actin.

Cytotoxicity and IFNy ELISA

Parental 3T3 and 3T3.FAP cells were transduced with luciferase aspreviously described (Moon et al., 2011, Clin. Cancer Res. 17:4719-30.Cytotoxicity assays were performed by co-culture of T cells with target3T3 cells at the indicated ratios, in triplicate, in 96-well roundbottom plates. After 18 hours, the culture supernatants were collectedfor IFNy analysis using an ELISA (mouse IFNy, BD OpEIA). Cytotoxicity oftransduced T cells was determined by detecting the remaining luciferaseactivity from the cell lysate using as assay described in Riese et al.,2013, Cancer Res. 73:3566-77.

CAR-T Cell Transfer into Mice Bearing Established Tumors

Mice were injected subcutaneously with 2×10⁶ AE17.ova (C57BL/6 mice),1×10⁶ TC1 (C57BL/6 mice), 2×10⁶ LKR (C57BL/6 crossed with 129 pf/j),0.5×10⁶ 4T1 (BALB/c mice), or 1×10⁶ CT26 (BALB/c mice) tumor cells intothe dorsal-lateral flank. Tumor (100-150 mm³)-bearing mice were randomlyassigned to receive either FAP-CAR T cells or MigR1-transduced T cellsor remained untreated (minimum, five mice per group, each experimentrepeated at least once). 1×10⁷ T cells were administered through thetail vein. Body weight and the tumor size were measured by electronicscales and calipers, respectively. At the end of the experiment, tumorsand spleens were harvested for flow cytometric analyses.

Flow Cytometric Analyses

Tumors were harvested 3 and 8 days after adoptive transfer of FAP-CAR Tcells to analyze intratumoral cells by flow cytometry (Zhao et al.,2010, Cancer Res. 70:9053-61. Cell acquisition was performed on LSR-IIusing FACSDiva software (BD Bioscience, USA). Data were analyzed usingFlowJo (Tree Star).

Statistical Analyses

For flank tumor studies comparing two groups, the student t test wasused. For comparisons of more than two groups, we used one-way ANOVAwith appropriate post hoc testing. Differences were consideredsignificant when p<0.05. Data are presented as mean+/−SEM.

The Results of the experiments disclosed in this Example are nowdescribed.

In Vitro Evaluation of Mouse FAP-CAR T Cells

The primary retroviral CAR construct (containing the scFv fromanti-murine FAP antibody 73.3 coupled to the human CD3Z and 4-1BBcytoplasmic domains and a control virus expressing only GFP (FIGS.11A-11D) were used to transduce activated mouse T cells resulting ingreater than 60% of T cells expressing GFP (MigR1) or GFP plus FAP-CAR(FIG. 12A).

To verify functionality, mouse T cells expressing FAP-CAR werestimulated for 18 hours with beads coated with either bovine serumalbumin (BSA) (negative control), recombinant FAP protein, oranti-CD3/anti-CD28 antibodies (positive control). The FAP-coated beadsactivated FAP-CAR T cells to increase CD69 expression, however, asexpected, to lower levels than that of CD3/CD28. (FIG. 12B).

To further evaluate intracellular signaling, lysates frombead-stimulated T cells were run on gels and immunoblotted. Incomparison to BSA-coated beads, FAP-coated beads induced phosphorylationof AKT, ERK, and IKKa/p in FAP-CAR T cells (FIG. 13).

To assess effector functions, transduced mouse T cells were co-culturedwith 3T3 fibroblasts (which do not express FAP) or with 3T3 fibroblaststransduced to express FAP (3T3.FAP) (FIGS. 9A and 9B). After 18 hours, Tcells expressing the FAP-CAR construct (but not the controlGFP-expressing T cells) effectively killed 3T3.FAP fibroblasts (FIG.12C) and secreted IFNy (FIG. 12D) in a dose-dependent manner, but had noeffect on parental 3T3 cells.

Injection of Mouse FAP-CAR T Cells Reduces Tumor Growth in aFAP-Specific Fashion

The capability of FAP-CAR mouse T cells to inhibit growth of tumors wasexplored using three different tumor lines which do not express FAP(FIG. 9C-9E): AE17.ova mesothelioma cells, TC1 and LKR lung cancercells. Cells were injected into the flanks of syngenic mice and allowedto form established tumors. The tumors had an easily detectable numberof mouse FAP-expressing cells with the majority of the FAP+ cells beingCD45⁻/CD90+ stromal cells (˜3% of total tumor cells), and only a smallminority being CD45+ hematopoietic cells (˜0.2% of total tumor cells)(FIG. 20 and FIG. 14).

When tumors reached ˜100-200 mm^(3t) (10-14 days after tumor cellinoculation), 10⁷ T cells were injected intravenously and tumormeasurements made serially. FAP-CAR T cells, but not MigR1 T cells,significantly (p<0.05) reduced the growth of TC1 tumors (FIG. 15A), LKRtumors (FIG. 15B) and AE17.ova tumors (FIG. 15C) by 35-50%.

To confirm specificity, AE17.ova cells were inoculated into FAP-nullC57BL/6 mice and the tumors were treated as above. In contrast to theeffect on AE17.ova tumors in wild-type C57BL/6 mice (FIG. 15C), FAP-CART cells had no effect on the growth of AE17.ova tumors in FAP-null mice(FIG. 15D). Given the differences between the efficacy data presentedherein and that of Tran et al. (2013, J Exp Med 210:1125-35, two of thesame tumor lines, CT26 and 4T1, that they reported were also treated. Incontrast to their findings, the FAP-CAR construct of the presentinvention induced significant reduction in tumor size (FIGS. 16A-16B),although the changes were smaller than those seen in FIGS. 15A-15D.

Effect of the Injection of Mouse FAP-CAR T Cells on FAP+ Cells

To evaluate the effect of the T cells on the FAP+ stromal cells, tumorswere harvested 7 to 9 days post-T cell infusion and the dissociatedcells were analyzed by flow cytometry. As shown in FIG. 20, at this timepoint, the FAP+/CD45⁻/CD90+ and FAP+/CD45+ populations were decreased byabout 50% in comparison with the untreated group, while the amount ofFAP+ cells remaining in MigR1 group was similar to the untreatedcontrols.

The AE17.ova model was chosen to characterize this depletion in moredetail, and the FAP+ cells were evaluated at 3 days post-T celltransfer. At this earlier time point, larger decreases in FAP+CD90+stromal cells (82%) and FAP+CD45+ leukocytes (56%) were observed (FIG.17A). Moreover, both low- and high-FAP expressing cells in both theCD45⁻CD90+ and CD45+ populations could be identified (FIG. 14). Whenthese specific populations were gated on, it was observed that FAP-CAR Tcells selectively depleted the FAP-high expressing cells, with littleeffect on FAP-low expressing cells (FIGS. 17B and 17C).

Kinetics of FAP-CAR T Cell Persistence

The number of intratumoral FAP-CAR T cells in the AE17.ova model wasassessed at 3, 7, and 10 days after adoptive transfer. It was discoveredthat the number peaked at day 3 after injection and diminished at the 7and 10 day time points by ˜65% (FIG. 18A).

To determine if this rapid loss of T cells was a consequence of abnormalfunction of the human CD3Z and 4-1BB cytoplasmic domains within mouse Tcells, a second construct was engineered by inserting the scFv anti-FAP73.3 antibody fragment into a fully murine CAR containing the murineCD3Z chain and the murine CD28 cytoplasmic domain (73.3m28z) (FIG. 11D).In vitro, this construct exhibited similar cytotoxicity and IFN-yrelease when reacted with FAP-expressing fibroblasts compared to the“human” version of CAR (FIGS. 19A-19C).

After injection into mice bearing AE17.ova tumors, the trafficking andpersistence of the two types of FAP-CAR T cells were observed to besimilar (FIG. 18B), as was antitumor efficacy (FIG. 18C). These datashow that, compared to human CAR, mouse CAR T cells have a shortpersistence time, despite the additional human or mouse co-stimulatorycytoplasmic domains.

Approaches to Enhance FAP-CAR T Cell Therapy

Since the T cells persist for only short periods of time in vivo, it washypothesized that giving a second infusion of FAP-CAR T cells wouldenhance therapeutic efficacy. AE17.ova tumor cells were injected intoflanks of C57BL/6 mice. When tumors reached approximately 100 mm³, afirst dose of FAP-CAR T cells was given intravenously. One week later,we randomly divided the FAP-CAR-treated animals were randomly dividedinto two groups, one treated with an additional dose of MigR1 (Singledose, FIG. 21A) and one treated with a second dose of FAP-CAR T cells(Double dose, FIG. 21A). At two weeks, tumors in the mice given twodoses of FAP-CAR T cells were significantly smaller (p<0.05) than thosein mice given only one dose of FAP-CAR T cells.

The efficacy of comparably transduced FAP-CAR splenic T cells isolatedfrom WT C57BL/6 versus DGK^-null mice was compared. DGKZ knockoutFAP-CAR T cells were more efficient in lysing 3T3.FAP cells (FIG. 22A)and in secreting IFNy (FIG. 22B) with retention of specificity in vitro.The DGK^-deficient FAP-CAR T cells were also more efficient (p<0.05 onday 11) after being injected into AE17.ova bearing mice (FIG. 21B). Theincreased efficacy was associated with greater persistence of theDGK^-knockout compared to WT FAP-CAR T cells (GFP+ cells) (FIG. 23).Thus, the enhanced anti-tumor efficacy was likely due to both increasedT cell activity and to increased persistence.

Role of the Acquired Immune System in the Efficacy of FAP-CAR T Cells

The role of the acquired immune system in the FAP-CAR T cell-mediatedanti-tumor response was evaluated by injecting AE17.ova tumors into theflanks of wild-type C57BL/6 mice or in immunodeficient NSG mice andtreating with one injection of 10⁷ FAP-CAR T cells. AE17.ova tumors grewmore rapidly in NSG than wild-type mice (FIG. 24A vs 24B) reflecting theendogenous anti-tumor activity in wild-type mice that was lost in theNSG mice. In contrast to the efficacy of the mouse FAP-CAR T cells inwild-type mice (FIG. 24A), the mouse FAP-CAR T cells had no anti-tumoreffects on the AE17.ova tumors in the immunodeficient NSG mice, (FIG.24B). This loss in activity was not due to loss of FAP expression in theNSG tumor microenvironment, as we confirmed that AE17.ova tumor in NSGmice develop a similar level of FAP expression as in immune-competentC57BL/6 mice.

To further explore this issue, taking advantage of the fact that TC1tumor cells express the viral oncogenic protein HPV-E7 and that AE17.ovacells express chicken ovalbumin, the impact of FAP⁺ cell depletion onendogenous anti-tumor immunity using E7- or ova-specific tetramerstaining of the infiltrating lymphocytes 8 days after adoptive transferwas evaluated. A significantly (p=0.02) increased percentage of totalCD8⁺ T cells within the tumors of FAP-CAR-treated mice compared tocontrol or MigR1-T cell-treated mice was observed (FIGS. 24C and 24E).In addition, tumors from FAP-CAR-treated mice had significantly(p=0.015) increased numbers of E7-specific T cells (FIG. 24D) orova-specific T cells within the TC-1 and AE17.ova tumors respectively(FIG. 24F).

To better understand the mechanisms of this immune response, the aboveexperiment was repeated with AE17.ova tumor-bearing mice, but theendogenous (non-GFP-expressing) T cells were analyzed at 3 and 8 daysafter T cell injection. Consistent with previous findings by Kraman etal. (2010, Science 330:827-30) who used a genetic approach to ablateFAP+ cells, the number of intratumoral T cells was similar between allthree groups at 3 days post adoptive transfer (FIG. 25A). However, atthis time point, the number of CD4+ T cells producing TNFa wassignificantly higher in FAP-CAR T cell group compared to untreated andcontrol T cell groups (FIG. 26A; black bars), while there was nodifference in the numbers of CD69+ and 4-1BB+ T cells, nor inIFN-y-producing CD8 T cells (FIGS. 26B-26D; black bars). At 8 daysfollowing treatment of FAP-CAR T cells, the number of T cells was higherin tumors treated with FAP-CAR T cells (as above) compared to the twocontrol groups (FIG. 25B). At this time point, however, the number ofCD69+ and IFNy+ CD8+ T cells was increased (FIGS. 26B and 26D; graybars), while TNF producing T cells and 4-1BB+ expressing T cells weresimilar among the groups (FIGS. 26A and 26C; gray bars). Together, theseresults establish that depletion of FAP+ cells in tumors enhancesenhance anti-tumor immunity by initially activating endogenous T cells,followed by increasing intra-tumor T cell infiltration at a latertimepoint.

Augmentation of the Efficacy of FAP-CAR T Cells by Combination with anAnti-Tumor Vaccine

Given the effects of FAP+ tumor cell depletion on anti-tumor immunity,it was hypothesized that combining a tumor vaccine with FAP-CAR T celladministration would enhance anti-tumor efficacy compared to eitherapproach alone. The HPV-E7-expressing TC1 tumor cells were injectedsubcutaneously into C57BL/6 mice, when tumors reached approximately 200mm³ saline or one subcutaneous dose of a vaccine consisting of 10⁹ pfuof an adenovirus expressing HPV-E7 (Ad.E7) (black arrow) wasadministered to boost the endogenous T cell response againstE7-expressing cells. Four days after saline injection or vaccination,FAP-CAR T cells (10⁷ cells; gray arrow) were given intravenously. Boththe Ad.E7 cancer vaccine and the FAP-CAR T cells, had only modesteffects on these large established tumors by themselves (FIG. 21C).However, the combination was able to induce tumor regression and inhibittumor growth up to two weeks before tumors started progressing.

Toxicity

Since FAP is an endogenous protein and toxicity (especially weight lossand anemia) was recently reported after depletion of FAP+ cells eitherby genetic ablation or FAP-CAR T cell administration, possibleoff-tumor/on-target adverse effects after administration of our FAP-CART cells were assessed. No clinical toxicity or anemia in any of theFAP-CAR T cell studies described herein was observed. The body weight oftumor-bearing mice remained the same or increased throughout eachexperiment (FIGS. 27A-27F).

To further evaluate toxicity, necropsies were performed and visceralorgans (heart, lungs, pancreas, liver, spleen, kidneys, skeletal muscle,and bone marrow) were harvested, sectioned, stained and analyzed in ablinded fashion eight days after T cell injection in mice treated withone dose of WT FAP-CAR T cells and eight days after a second dose ofWT-FAP CAR T cells from the mice from the experiment shown in FIG. 21A.When compared to control tumor-bearing mice, no abnormalities wereobserved in the mice given WT FAP-CAR T cells. This specificallyincluded lack of bone marrow hypoplasia (FIGS. 28A-28C) or any change inskeletal muscle.

Necropsies on the mice 8 days after injection of the hyperactiveDGK^-deficient FAP-CAR T cells from the experiment depicted in FIG. 21B.No abnormalities were noted, except in the pancreatic sections thatexhibited some mild focal peri-vascular and peri-islet lymphocyticinfiltration (FIG. 29C). These changes were not seen in mice injectedwith wild-type FAP-CAR T cells (FIG. 29B).

The anti-tumor efficacy and safety of chimeric antigenreceptor-transduced T cells targeted to cells expressing FAP, a targetthat is highly up regulated in tumor stroma has been investigatedherein. Given that cancer-associated stromal cells appear to have amajor immune modulating effect on both innate and acquired immunity, itwas important to use fully immune-competent mice, without ablation ofbone marrow cells, so that the role of the acquired immune system inFAP-CAR T cell-mediated anti-tumor response could be evaluated.

The data presented herein establish that mouse FAP-CAR T cells exhibitantigen-specific cytotoxicity against FAP+ stromal cells and markedlyreduce the rare subset of FAP+/CD45+/F4/80+ myeloid cells and the moreprevalent FAP+/CD90+ stromal cells detected in multiple mouse models ofestablished mesothelioma and lung cancer (FIG. 20). A single treatmentwith FAP-CAR T cells resulted in ˜80% depletion of the FAP^(hi) stromalcells at 3 days following treatment of FAP-CAR T cells (FIG. 17A),leaving white blood cells and FAP^(IQ) cells relatively unaffected(FIGS. 17B and 17C). The depletion of FAP+ cells was associated with asignificant inhibition (35-50%) of tumor growth compared to untreatedand vector control-transduced CAR T cell (MigR1) treated tumors (FIG.15A-15C, FIGS. 16A-16B). Importantly, the anti-tumor activity of FAP-CART cells was lost in FAP-null mice (FIG. 15D) indicating that theanti-tumor activity of FAP-CAR T cells is dependent on expression of FAPon host-derived cells.

It has also been discovered in the present invention that the anti-tumorefficacy of FAP-CAR T cells was lost in immunodeficient mice (FIG. 24B)emphasizing the importance of the acquired immune system at least in thetumor models employed in this study. To more completely understand thiseffect, the endogenous T cells within the tumors were evaluated at 3 and8 days after CAR-T cell infusion. At the earlier time-point, no increasein T cell infiltration or CD8 T cell activation was observed. However,an increase in the number of CD4+ T cells producing TNF-α was evident(FIG. 26A). At the later time-point, an increase in infiltration oftotal CD8+ T cells within the tumors was observed, as well asantigen-specific CD8+ T cells in both the AE17.ova and E7-positive TC1tumors. More IFNy producing CD8+ T cells and more CD69+ T cells werealso found at this time (FIGS. 26B and 26D). These data suggest thatFAP-CAR T cells enter the tumors and deplete FAP+ cells, which throughan unknown mechanism activates endogenous CD4+ T cells to produce TNF.The high levels of TNF may induce tumor cell apoptosis, as well asinduce a temporary tumor vasculature shut down which may limit earlyinfiltration by endogenous T cells. It appears that by 8 days afterFAP-CAR T cell treatment, activated CD8+ cells enter the tumor andfunction to further limit tumor growth. However, it should be noted thatin mouse tumors, which are relatively immunogenic tumors and haverelatively few fibroblasts, the contribution of the immune-mediatedmechanisms may be relatively prominent compared to the potentialcontribution of non-immune mediated mechanisms (i.e. alterations inmatrix and/or angiogenesis) that might be seen in non-immunogenic, morefibroblast-rich tumors. Preliminary studies using more desmoplastic,non-immunogenic mouse tumor models and human xenografts support thisidea.

In the models presented herein, the use of FAP-CAR T cells led to asignificant, but only temporary inhibition of tumor growth. One reasonfor the transience of the effect is likely the relatively shortpersistence of the murine CAR T cells; the number of intra-tumoralmurine FAP-CAR T cells rapidly decreased with time (FIG. 18A). This islikely due to a number of well-known intrinsic differences between mouseand human T cells. After expanding human CAR T cells, the lymphocytescan be “rested down” before injection and are less sensitive toimmediate activation-induced cell death (AICD). In the mouse system, thecells are highly activated (needed for retroviral transduction) wheninjected. Whereas the human T cells persist and proliferate in tumorsfor weeks, many of the transduced mouse T cells undergo AICD (FIGS.30A-30B) and have a relatively short lifespan compared to human cells.To ensure this short lifespan was not due to the fact that the constructused herein incorporated human CD3Z and human 4-1BB activation domains,a fully mouse FAP-CAR construct was made that had the 73.3 scFv coupledwith the mouse CD3Z chain and mouse CD28 domain (FIG. 11D). Virtuallyequal efficacy in killing, cytokine production, persistence, as well asanti-tumor activity between T cells expressing the two constructs wasobserved (FIGS. 19A-19C, FIG. 18D). In addition, both types of CARs wereequally susceptible to ACID (FIG. 30B). FAP-CAR mouse T cells wereexamined to see if they would be enhanced if they were “preconditioned”in the host by inducing lymphodepletion or IL-2 administration, but noincrease in efficacy in mice irradiated prior to injection of ourFAP-CAR T cells was observed.

Given the lack of persistence of the FAP-CAR T cells (FIG. 18A), it wasnot surprising that efficacy could be enhanced by giving a second doseof T cells one week after the first (FIG. 21A), clearly demonstratingthat enhanced persistence could augment efficacy. Also, by blocking a Tcell-intrinsic negative regulatory mechanism (up-regulation of theenzyme DGK), the killing ability and persistence of murine CAR T cellscould be augmented. In addition, FAP-CAR T cells deficient in DGKZshowed enhanced ability to kill FAP expressing cells in vitro and wereclearly more efficacious in vivo (FIG. 21B). These data thus suggestthat it will be advantageous to optimize both the persistence andpotency of the T cells.

Despite using different mouse strains in each mouse tumor model, lessthan 0.1% of FAP+ stromal cells in the lungs and bone marrows wereobserved. In contrast, 3-5% of dissociated cells from the pancreasexpressed FAP. However, when FAP expression on those pancreatic stromalcells was compared with the tumor-associated stromal cells isolated fromthe same hosts, cancer associated stromal cells expressed higher levelsof FAP (FIGS. 31A-31D).

Administration of one dose or even two doses of the FAP-CAR T cells didnot cause any weight loss (FIGS. 27A-27F), nor did they cause anydecrease in hematocrit or increase in serum amylase. Furthermore,detailed histologic analyses of necropsy samples showed no microscopicabnormalities, and specifically no damage to muscle, pancreas (FIGS.29A-29C), or bone marrow (FIGS. 28A-28C).

Thus, the data establish that it is possible to partially depletetumor-associated FAP⁺ cells while retaining anti-tumor efficacy, butwithout eliciting severe side effects. This may be related to the factthat our FAP-CAR T cells were able to efficiently eliminate the FAPwhile sparing the cells expressing lower levels of FAP (FIGS. 17A-17C),like those in the pancreas, and presumably like those in the bone marrowand muscle. The only instance in which any histologic abnormalititeswere observed was in the pancreas (FIG. 29C), but only when hyperactiveDGK^-deleted cells were used.

Anti-tumor activity with CARs containing either human and mousecytoplasmic domains was observed (FIG. 18C). The data suggest thatendogenous immune effects may be important for the efficacy of the CARs.

The data also establish that FAP-CAR T cells may be used in combinationwith other therapies. This can include combination with chemotherapywhere stromal disruption could enhance drug delivery and combinationswith immunotherapy. In the present invention, FAP-CAR T cells werecombined with an Ad.E7 cancer vaccine which generates E7-specificadaptive immune response against TC1 cells. As expected, neither thecancer vaccine nor FAP-CAR T cells worked well on large, establishedtumors (FIG. 21C). However, in combination, there was an additive, andperhaps synergistic effect against TC1 tumors.

In summary, the data establish that FAP-CAR T cells recognize targetcells in an antigen-specific manner and reduce tumor growth in vivo.Targeting tumor stromal cells with CAR T cells augmented anti-tumorimmunity. It is likely that he efficacy of FAP-CAR T cells can beenhanced by improving persistence of T cells, producing more highlyactive T cells, administering multiple doses of FAP-CAR-T cells, and bycombining with other types of immunotherapy or chemotherapy. Toxicitywas not observed, at least under dosing conditions tested herein inotherwise healthy tumor-bearing animals.

Additional Data

FAP-CAR T Cells are Efficacious in A Mouse Pancreatic Cancer Model andin an Autochthonous Mouse Lung Cancer Model

The efficacy of the FAP-CAR T cells in a pancreatic cancer model calledPDA4662 was tested. In comparison to other flank tumor models, thisparticular tumor model shows more desmoplastic characteristic, and isnon-immunogenic. 10⁷ anti-mouse FAP-CAR T cells or control T cells(MigR1) were injected into mice when PDA4662 tumors reached 100 mm³ insize. Tumor measurements were then followed. FAP-CAR T cells inducedsignificant inhibition in tumor growth, while the control T cells didnot have any effect on tumor growth (FIG. 32).

An autochthonous lung cancer mouse model was generated by crossingLSL-KrasG12D mice that carry an inducible constitutively active mutantallele of Kras, with conditional TGFBRIIflox/flox knock-out mice. Theoncogenic Kras mutation is relevant to human disease as Kras mutationand/or over expression is the most common oncogenic event associatedwith human lung cancer. The Kras/TGFBRII KO model, with simultaneousinduction of Kras and deletion of TGFβRII, leads to the development oftumors that more closely resemble primary human lung cancer with regardto invasion and collagen deposition. These animals die with largeinvasive lung tumors (and mediastinal metastases) within 4 weeks.Briefly, mice that were heterozygous for the LSL mutant KrasG12D geneand homozygous for a floxed TGBβRII gene were anesthetized and given 10⁹pfu of an adenovirus expressing Cre via the intranasal route to bothinduce tumors and inactivate the TGF-β receptor in the same epithelialcells. Two weeks after Ad.Cre injection, 10⁷ anti-mouse FAP-CAR murine Tcells or control murine T cells (MigR1) were administered intravenously.In total, three weekly doses of T cells were given to the mice. In FIG.33 the overall survival of those Kras/TGFbRII mice after adoptivetransfer of FAP-CAR T cells can be seen. FAP-CAR T cells, but notcontrol T cells, prolonged the lifespan of those mice with induciblelung cancer.

Evaluation of the Use of mRNA-Transduced CARs to Provide Short TermPersistence

No toxicity was noted in mice treated with virally-transduced FAP-CAR Tcells. However, it is important to provide a potential alternative,namely mRNA electroporation, for transient CAR expression. It washypothesized that delivery of T cells that have been electroporated withFAP-CAR mRNA would be efficacious and safe. To accomplish this, vectorsand electroporation techniques were developed that facilitatetransfection of human T cells with CAR mRNA and that achieve high-levelsurface expression for 3-5 days. The main rationale for this strategywas to have T cells that only transiently express the CAR as a keysafety feature. Any off-target effects mediated by CAR should belimited.

FIG. 34 illustrates high surface FAP-CAR expression in human T cellsthat were electroporated 48 hours earlier with anti-mouse FAP-CAR mRNA.The black line shows high level CAR expression in the electroporatedcells versus non-transfected cells and isotype control. These mRNAtransfected T cells can effectively and selectively kill the 3T3 cellline transduced with mouse FAP in vitro (FIG. 35—see purple line). Todemonstrate that the mRNA T cells have in vivo efficacy, on Day 14, 10⁷mRNA-transduced human T cells (FAP-CAR) or non-transduced T cells (NTD)were injected every week for two doses into immunodeficient mice bearingestablished human A549 tumors (which induce large amounts of FAP⁺ CAFs).FIG. 36 illustrates that these cells have clear anti-tumor efficacy.These new data establish that mRNA-transfected CAR T cells can functionefficiently in T cells and direct killing of tumor cells both in vitroand in an animal tumor model. There was no change in body weight andthere was no sign of anemia in those mRNA CAT T cells-treated mice.

Evaluation of Anti-Human FAP CAR T Cells

The sequence of the light and heavy chains of the well-characterized F19anti-human FAP monoclonal antibody was obtained from the LudwigInstitute.

These chains were inserted into the 4-1BB:CD3ζ “double activationdomain” construct and a lentivirus was produced. Human T cells weretransduced and evaluated with regard to expression level, and theability to secrete cytokines and kill human FAP-expressing cells.Untransduced T cells or the anti-huFAP-CAR T cells (labeled 1142 on thefigures) were reacted for 18 hours with either wild type (parental) 3T3pcells (mouse fibroblasts that do not express FAP) or 3T3 cellstransduced to express human FAP (3T3huFAP) at different T cell tofibroblast ratio. FIGS. 37 and 38 illustrate that huFAP T cellsselectively and effectively kill 3T3 cells expressing human FAP with noeffect of control T cells. FIGS. 39 and 40 illustrate that only huFAP Tcells selectively release IFNγ after exposure of 3T3 cells expressinghuman FAP. These data establish that the humanFAP-CAR T cellsselectively and efficiently release IFNγ and kill fibroblast cellsexpressing human FAP.

Safety of FAP-CAR T Cells in Wound Healing

Mice were given a calibrated skin wound on their backs using a dermalbiopsy punch using techniques described in the art. Four hours postwound induction, mice received either 10⁷ muFAP-CAR murine T cells or10⁷ retrovirally murine T cells transduced with the control CAR to ruleout non-specific effects. Another dose of T cells was adoptivelytransferred 3 days later. In single-blinded fashion, the wound size wasmeasured daily in each mouse and the groups compared. FIG. 41illustrates that FAP-CAR T cells did not affect the closure of wounds.In addition, FAP-CAR T cells did not cause weight loss or toxicity inpancreas, which was determined by measuring plasma amylase level (FIG.42).

Safety of FAP-CAR T Cells in Lung Fibrosis

FAP is expressed in the lungs of patients with pulmonary fibrosis.Although the “standard” model to study pulmonary fibrosis isinstillation of bleomycin, this model is actually highly inflammatoryand results in only transient fibrosis. A much better model of the humandisease is fibrosis secondary to lung irradiation.

C57/B16 mice (n=20 per group) received 13.5 Gy of chest irradiation. Atfour months post-irradiation, at a time when pulmonary fibrosis is knownto exist, mice were randomized to receive 10⁷ control CAR T cells or 10⁷anti-mouse FAP-CAR T cells. After one week, one set of mice weresacrificed. The remainder were given saline or a second dose of controlor FAP-CAR T cells and sacrificed after an additional week. Using FACSanalysis, increased FAP⁺ cells were seen in the lungs of controlirradiated mice (compared to non-radiated mice). These cells weresignificantly decreased after CAR treatment showing the T cells hadactivity (FIG. 43). Importantly, no toxicity was observed after eitherthe first or second infusion of FAP CAR T cells (specifically, noincreased deaths, nor changes in weight, oxygenation, or respirationrate was observed). Staining of the lungs and FACS analysis showed noevidence of increased inflammation in the lungs of FAP-CAR T cell group.These preliminary data support the safety of FAP-CAR T cells, even inthe presence of lung fibrosis.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. An isolated chimeric antigen receptor (CAR)comprising an antigen binding domain encoded by a nucleic acid sequencecomprising SEQ ID NO: 3, a transmembrane domain, a costimulatorysignaling region, and a CD3 zeta signaling domain, wherein the antigenbinding domain binds to a stromal cell antigen.
 2. The isolated CAR ofclaim 1, wherein the stromal cell antigen is expressed on a stromal cellpresent in a tumor microenvironment.
 3. The isolated CAR of claim 2,wherein the tumor is a carcinoma.
 4. The isolated CAR of claim 1,wherein the stromal cell antigen is fibroblast activation protein (FAP).5. The isolated CAR of claim 1, wherein the costimulatory signalingregion comprises the intracellular domain of a costimulatory moleculeselected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30,CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2,CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83,and any combination thereof.
 6. The isolated CAR of claim 1, wherein theCAR is encoded by a nucleic acid sequence comprising SEQ ID NO:
 1. 7.The isolated CAR of claim 1, wherein the transmembrane domain is encodedby a nucleic acid sequence comprising SEQ ID NO:
 5. 8. The isolated CARof claim 1, wherein the costimulatory signaling region is encoded by anucleic acid sequence comprising SEQ ID NO:
 6. 9. The isolated CAR ofclaim 1, wherein the CD3 zeta signaling domain is encoded by a nucleicacid sequence comprising SEQ ID NO: 7.